Journal articles: 'Isoform quantification'

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Chabbert, Christophe D., Tanja Eberhart, Ilaria Guccini, Wilhelm Krek, and Werner J. Kovacs. "Correction of gene model annotations improves isoform abundance estimates: the example of ketohexokinase (Khk)." F1000Research 7 (December 19, 2018): 1956. http://dx.doi.org/10.12688/f1000research.17082.1.

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Next generation sequencing protocols such as RNA-seq have made the genome wide characterization of the transcriptome a crucial part of many research projects in biology. Analyses of the resulting data provide key information on gene expression and in certain cases on exon or isoform usage. The emergence of transcript quantification software such as Salmon has enabled researchers to efficiently estimate isoform and gene expressions across the genome while tremendously reducing the necessary computational power. Although overall gene expression estimations were shown to be accurate, isoform expression quantifications appear to be a more challenging task. Low expression levels and uneven or insufficient coverage were reported as potential explanations for inconsistent estimates. Here, through the example of the ketohexokinase (Khk) gene in mouse, we demonstrate that the use of an incorrect gene annotation can also result in erroneous isoform quantification results. Manual correction of the input Khk gene model provided a much more accurate estimation of relative Khk isoform expression when compared to quantitative PCR (qPCR measurements). In particular, removal of an unexpressed retained intron and a proper adjustment of the 5’ and 3’ untranslated regions both had a strong impact on the correction of erroneous estimates. Finally, we observed a better concordance in isoform quantification between datasets and sequencing strategies when relying on the newly generated Khk annotations. These results highlight the importance of accurate gene models and annotations for correct isoform quantification and reassert the need for orthogonal methods of estimation of isoform expression to confirm important findings.

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Chabbert, Christophe D., Tanja Eberhart, Ilaria Guccini, Wilhelm Krek, and Werner J. Kovacs. "Correction of gene model annotations improves isoform abundance estimates: the example of ketohexokinase (Khk)." F1000Research 7 (April 3, 2019): 1956. http://dx.doi.org/10.12688/f1000research.17082.2.

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Next generation sequencing protocols such as RNA-seq have made the genome-wide characterization of the transcriptome a crucial part of many research projects in biology. Analyses of the resulting data provide key information on gene expression and in certain cases on exon or isoform usage. The emergence of transcript quantification software such as Salmon has enabled researchers to efficiently estimate isoform and gene expressions across the genome while tremendously reducing the necessary computational power. Although overall gene expression estimations were shown to be accurate, isoform expression quantifications appear to be a more challenging task. Low expression levels and uneven or insufficient coverage were reported as potential explanations for inconsistent estimates. Here, through the example of the ketohexokinase (Khk) gene in mouse, we demonstrate that the use of an incorrect gene annotation can also result in erroneous isoform quantification results. Manual correction of the input Khk gene model provided a much more accurate estimation of relative Khk isoform expression when compared to quantitative PCR (qPCR measurements). In particular, removal of an unexpressed retained intron and a proper adjustment of the 5’ and 3’ untranslated regions both had a strong impact on the correction of erroneous estimates. Finally, we observed a better concordance in isoform quantification between datasets and sequencing strategies when relying on the newly generated Khk annotations. These results highlight the importance of accurate gene models and annotations for correct isoform quantification and reassert the need for orthogonal methods of estimation of isoform expression to confirm important findings.

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Sun, Jiao, Jae-Woong Chang, Teng Zhang, Jeongsik Yong, Rui Kuang, and Wei Zhang. "Platform-integrated mRNA isoform quantification." Bioinformatics 36, no. 8 (December 13, 2019): 2466–73. http://dx.doi.org/10.1093/bioinformatics/btz932.

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Abstract Motivation Accurate estimation of transcript isoform abundance is critical for downstream transcriptome analyses and can lead to precise molecular mechanisms for understanding complex human diseases, like cancer. Simplex mRNA Sequencing (RNA-Seq) based isoform quantification approaches are facing the challenges of inherent sampling bias and unidentifiable read origins. A large-scale experiment shows that the consistency between RNA-Seq and other mRNA quantification platforms is relatively low at the isoform level compared to the gene level. In this project, we developed a platform-integrated model for transcript quantification (IntMTQ) to improve the performance of RNA-Seq on isoform expression estimation. IntMTQ, which benefits from the mRNA expressions reported by the other platforms, provides more precise RNA-Seq-based isoform quantification and leads to more accurate molecular signatures for disease phenotype prediction. Results In the experiments to assess the quality of isoform expression estimated by IntMTQ, we designed three tasks for clustering and classification of 46 cancer cell lines with four different mRNA quantification platforms, including newly developed NanoString’s nCounter technology. The results demonstrate that the isoform expressions learned by IntMTQ consistently provide more and better molecular features for downstream analyses compared with five baseline algorithms which consider RNA-Seq data only. An independent RT-qPCR experiment on seven genes in twelve cancer cell lines showed that the IntMTQ improved overall transcript quantification. The platform-integrated algorithms could be applied to large-scale cancer studies, such as The Cancer Genome Atlas (TCGA), with both RNA-Seq and array-based platforms available. Availability and implementation Source code is available at: https://github.com/CompbioLabUcf/IntMTQ. Supplementary information Supplementary data are available at Bioinformatics online.

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Abendschein, D. R., H. L. Fontanet, and R. Nohara. "Optimized preservation of isoforms of creatine kinase MM isoenzyme in plasma specimens and their rapid quantification by semi-automated chromatofocusing." Clinical Chemistry 36, no. 5 (May 1, 1990): 723–27. http://dx.doi.org/10.1093/clinchem/36.5.723.

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Abstract We report a convenient chromatofocusing procedure for rapid and sensitive quantification of isoforms of the MM isoenzyme of creatine kinase (EC 2.7.3.2) in plasma and efficient methods for preserving isoform profiles during handling of specimens. The assay involves use of prepacked, re-usable Mono P chromatofocusing columns and a "Fast Protein Liquid Chromatograph" (FPLC) system with on-line detection of isoform enzymatic activity in column effluent. Profiles of isoforms are analyzed within 25 min with the use of a 1-mL column; the lower limit of sensitivity for CK activity is 5 mU, and recovery of each isoform is within 1% of the amount added to plasma. Collection of blood specimens in Vacutainer Tubes containing 28.5 mumol of EDTA (final concentration in plasma, 7 to 10 mmol/L) inhibited carboxypeptidase activity in plasma by 76%, sufficient to essentially abolish isoform conversion in vitro at room temperature. These methods should facilitate applications of isoform analysis for diagnosis of myocardial infarction and coronary artery recanalization.

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Baines, I. C., A. Corigliano-Murphy, and E. D. Korn. "Quantification and localization of phosphorylated myosin I isoforms in Acanthamoeba castellanii." Journal of Cell Biology 130, no. 3 (August 1, 1995): 591–603. http://dx.doi.org/10.1083/jcb.130.3.591.

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The actin-activated Mg(2+)-ATPase activities of the three myosin I isoforms in Acanthamoeba castellanii are significantly expressed only after phosphorylation of a single site in the myosin I heavy chain. Synthetic phosphorylated and unphosphorylated peptides corresponding to the phosphorylation site sequences, which differ for the three myosin I isoforms, were used to raise isoform-specific antibodies that recognized only the phosphorylated myosin I or the total myosin I isoform (phosphorylated and unphosphorylated), respectively. With these antisera, the amounts of total and phosphorylated isoform were quantified, the phosphomyosin I isoforms localized, and the compartmental distribution of the phosphomyosin isoforms determined. Myosin IA, which was almost entirely in the actin-rich cortex, was 70-100% phosphorylated and particularly enriched under phagocytic cups. Myosins IB and IC were predominantly associated with plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmic myosins IB and IC, like cytoplasmic myosin IA, were mostly phosphorylated (60-100%). Moreover, phosphomyosin IB was concentrated in actively motile regions of the plasma membrane. More than 20-fold more phosphomyosin IC and 10-fold more F-actin were associated with the membranes of contracting contractile vacuoles (CV) than of filling CVs. As the total amount of CV-associated myosin IC remained constant, it must be phosphorylated at the start of CV contraction. These data extend previous proposals for the specific functions of myosin I isozymes in Acanthamoeba (Baines, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell Biol. 119: 1193-1203): phosphomyosin IA in phagocytosis, phosphomyosin IB in phagocytosis and pinocytosis, and phosphomyosin IC in contraction of the CV.

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Mahlapuu, Margit, Carina Johansson, Kerstin Lindgren, Göran Hjälm, Brian R. Barnes, Anna Krook, Juleen R. Zierath, Leif Andersson, and Stefan Marklund. "Expression profiling of the γ-subunit isoforms of AMP-activated protein kinase suggests a major role for γ3 in white skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 286, no. 2 (February 2004): E194—E200. http://dx.doi.org/10.1152/ajpendo.00147.2003.

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Expression patterns of the three isoforms of the regulatory γ-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The γ3-isoform appeared highly specific to skeletal muscle, whereas γ1 and γ2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of γ3. In samples of white skeletal muscle, γ3 clearly appeared to be the most abundant γ-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of γ3 mRNA expression, whereas levels of γ1 and γ2 remained largely unchanged. However, even in these cultured myotubes, γ2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a γ3-specific antibody showed that γ3 was exclusively associated with the α2- and β2-subunit isoforms. The observation that the AMPKγ3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant γ-isoform, strongly suggests that γ3 has a key role in this tissue.

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Yang, Sheng, Fang Shao, Weiwei Duan, Yang Zhao, and Feng Chen. "Variance component testing for identifying differentially expressed genes in RNA-seq data." PeerJ 5 (September 8, 2017): e3797. http://dx.doi.org/10.7717/peerj.3797.

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RNA sequencing (RNA-Seq) enables the measurement and comparison of gene expression with isoform-level quantification. Differences in the effect of each isoform may make traditional methods, which aggregate isoforms, ineffective. Here, we introduce a variance component-based test that can jointly test multiple isoforms of one gene to identify differentially expressed (DE) genes, especially those with isoforms that have differential effects. We model isoform-level expression data from RNA-Seq using a negative binomial distribution and consider the baseline abundance of isoforms and their effects as two random terms. Our approach tests the global null hypothesis of no difference in any of the isoforms. The null distribution of the derived score statistic is investigated using empirical and theoretical methods. The results of simulations suggest that the performance of the proposed set test is superior to that of traditional algorithms and almost reaches optimal power when the variance of covariates is large. This method is also applied to analyze real data. Our algorithm, as a supplement to traditional algorithms, is superior at selecting DE genes with sparse or opposite effects for isoforms.

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Mageean, Craig J., John R. Griffiths, Duncan L. Smith, Michael J. Clague, and Ian A. Prior. "Absolute Quantification of Endogenous Ras Isoform Abundance." PLOS ONE 10, no. 11 (November 11, 2015): e0142674. http://dx.doi.org/10.1371/journal.pone.0142674.

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Hohenegger, M. A., L. Fineder, F. Kronenberg, A. Lingenhel, A. Gruber, G. Utermann, and H. Dieplinger. "Apolipoprotein(α) isoform-independent quantification of lipoprotein(α)." Atherosclerosis 144 (May 1999): 109. http://dx.doi.org/10.1016/s0021-9150(99)80422-5.

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Hiller, David, and Wing Hung Wong. "Simultaneous Isoform Discovery and Quantification from RNA-Seq." Statistics in Biosciences 5, no. 1 (June 14, 2012): 100–118. http://dx.doi.org/10.1007/s12561-012-9069-2.

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Ikuta, Katsuya, Takaaki Hosoki, Yasushi Shimonaka, Yusuke Sasaki, Hideyuki Yasuno, Takaaki Ohtake, Katsunori Sasaki, Yoshihiro Torimoto, Keiji Saito, and Yutaka Kohgo. "Diferric Transferrin-Sensed Hepcidin Upregulation in Human Hepatoma-Derived Cell Line Is Differentially Controlled in Each Isoform 20, 22, 25; Confirmation by a Novel Simultaneous Quantification by Liquid Chromatography/ Tandem Mass Spectrometry." Blood 114, no. 22 (November 20, 2009): 4046. http://dx.doi.org/10.1182/blood.v114.22.4046.4046.

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Abstract Abstract 4046 Poster Board III-981 Introduction and aim Hepcidin is a key molecule of body iron metabolism, and the expression at mRNA level is thought to be upregulated by iron loading. As the mature processed form of human hepcidin is known to have 3 isoforms, hepcidin -20, -22, and -25, and hepcidin -25 is thought to be the major isoform active in iron metabolism. However, the physiological roles of other isoforms are poorly understood. Concerning the study on the regulatory mechanism on hepcidin expression, most studies have been only performed at the transcriptional level because of the difficulty of quantification of hepcidin in cell culture media; therefore, the experiments in vitro would be valuable. We therefore developed a sensitive new method for measuring hepcidin that can simultaneously measure the isoforms in culture media, and studied the expression patterns of isoforms at mature protein level in various human hepatoma-derived cell lines with and without diferric transferrin. Methods Quantification of human hepcidin -20, -22, -25 was performed using liquid chromatography (LC) - tandem mass spectrometry (MS) which we newly developed. Selected reaction monitoring (SRM) transitions and the collision energies were settled for each isoform respectively. Quantification of hepcidin isoforms in culture medium of 13 strains of hepatoma-derived cell lines was performed. Various stimulants for hepcidin expression, such as interleukin-6, diferric transferrin and etc, were also used for investigating the response patterns of hepcidin isoforms. Results Upon optimization of SRM conditions, the most intense precursor ions were selected in each mass spectrum to detect hepcidin isoforms. Product ions were selected to maximize sensitivity and selectivity. Despite using culture media including 10% FBS as matrix, isoform peaks were not interfered with by a blank matrix, indicating the method has good selectivity. Calibration curves were constructed over the range 2-1,000 ng/mL, and linearity of the calibration curves by weighted (1/x2) linear regression was excellent (correlation coefficient: r=0.9974 for hepcidin-20, r=0.9937 for hepcidin-22, r=0.9950 for hepcidin-25). Accuracies for back-corrected concentrations were 99.7-122.1% for hepcidin-20, 102.6-132.5% for hepcidin-22, and 99.1-141.2% for hepcidin-25. These results indicate that the method is adequate for quantifying hepcidin isoforms in culture media. We also found that substantial difference of hepcidin isoforms' expression patterns among human hepatoma-derived cell lines, and the patterns were divided into 5 groups. Response patterns for various stimulants were also different among those groups. Especially, human diferric transferrin upregulates hepcidin-20 and -22 in WRL68 cells, and hepcidin-22 in Hep3B, HuH-2, HuH-4, and HuH-6 cells; this should be the first report that human diferric transferrin upregulates hepcidin isoforms other than hepcidin-25 in human hepatocyte-derived cells. Conclusions We have devised a novel method for simultaneous quantification of hepcidin isoforms in culture media. Although most previous studies only observe the changes of hepcidin expression at mRNA level, our method revealed heterogeneous expressions of hepcidin isoforms and hepcidin upregulation by human diferric transferrin in human hepatocyte-derived cells at the peptide level. The fact of hepcidin isoforms' upregulation by human diferric transferrin in human hepatocyte-derived cells might be the clue to elucidate the mechanism for iron sensor in human body. We believe that this novel quantification method can contribute to further progress, especially in vitro research on the regulation of hepcidin expression. Disclosures: No relevant conflicts of interest to declare.

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Léguillette, Renaud, Fulvio R. Gil, Nedjma Zitouni, Stéphane Lajoie-Kadoch, Apolinary Sobieszek, and Anne-Marie Lauzon. "(+)Insert smooth muscle myosin heavy chain (SM-B) isoform expression in human tissues." American Journal of Physiology-Cell Physiology 289, no. 5 (November 2005): C1277—C1285. http://dx.doi.org/10.1152/ajpcell.00244.2004.

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Two smooth muscle myosin heavy chain isoforms differ in their amino terminus by the presence [(+)insert] or absence [(−)insert] of a seven-amino acid insert. Animal studies show that the (+)insert isoform is predominantly expressed in rapidly contracting phasic muscle and the (−)insert isoform is mostly found in slowly contracting tonic muscle. The expression of the (+)insert isoform has never been demonstrated in human smooth muscle. We hypothesized that the (+)insert isoform is present in humans and that its expression is commensurate with the organ's functional requirements. We report, for the first time, the sequence of the human (+)insert isoform and quantification of its expression by real-time PCR and Western blot analysis in a panel of human organs. The (+)insert isoform mRNA and protein expression levels are significantly greater in small intestine compared with all organs studied except for trachea and are significantly greater in trachea compared with uterus and aorta. To assess the functional significance of this differential myosin isoform expression between organs, we measured the rate of actin filament movement (νmax) when propelled by myosin purified from rat organs, because the rat and human inserts are identical and their remaining sequences show 93% identity. νmax exhibits a rank correlation from the most tonic to the most phasic organ. The selective expression of the (+)insert isoform observed among human organs suggests that it is an important determinant of tissue shortening velocity. A differential expression of the (+)insert isoform could also account for altered contractile properties observed in human pathology.

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Kataoka, Itaru, Fumihiko Ishimaru, Koichi Ichimura, Tadashi Yoshino, and Mitsune Tanimoto. "Aberrant Expression of Ikaros Induces T-Cell Leukemia/Lymphoma in Mice without Wild-Type Allelic Loss." Blood 106, no. 11 (November 16, 2005): 1224. http://dx.doi.org/10.1182/blood.v106.11.1224.1224.

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Abstract The Ikaros gene consisting of seven exons codes a family member of zinc finger transcription factors essential for establishment of immune systems. A number of isoform proteins are produced by alternative splicing. The activity of Ikaros is strictly regulated by total expression level or mutual dimerization with full-length and dominant-negative isoforms. Findings from Ikaros mutant mice revealed its characteristics as tumor suppressor in addition to functions of hemato-lymphoid development and differentiation. Mutant mice homozygous for lack of dimerization domains evolve T-cell leukemia and lymphoma. Heterozygous mutation of DNA binding domains can also lead to clonal expansion of T-cells through loss of heterozygosity. In both of murine models reported earlier, leukemias arise from the absence of full-length active isoforms. On the other hand, analyses for human diseases conducted by our group elucidated high incidence of over-expression of dominant-negative isoform in adult precursor B-lymphoblastic leukemia. Thus the mechanisms by which Ikaros contribute to leukemogenesis are critical issues to be clarified especially in human. Here, we demonstrate that over-expression of dominant-negative isoform causes leukemia and lymphoma without wild-type allelic loss by murine BMT assay. Retroviral gene transfer of dominant-negative isoform, Ik6, to murine hematopoietic cells developed lethal lympho-proliferative disorders in 3–6 months of latency with complete penetrantion. The diseases involved a variety of organs such as thymus, lymphnode, spleen, liver and bone marrow. Clonality of the infiltrating cells was confirmed by Southern blotting. The leukemic cells showed T-cell phenotype (B220-CD3e+CD4+CD8a+TCRb+Gr1-Mac1-) and express simultaneously full-length Ikaros proteins and transduced dominant-negative isoform. These data suggest that leukemogenesis mediated by decreased activity of Ikaros does not require deletion of full-length isoforms, and disproportional expression of Ikaros directed to dominant-negative isoform is enough to accumulate and immortalize lymphoid blasts in vivo. Silencing dominant-negative Ikaros proteins should be considered as a good candidate of molecular targeted therapy, and quantification of Ikaros isoforms might be useful for monitoring the minimal residual disease.

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Wu, K. D., W. S. Lee, J. Wey, D. Bungard, and J. Lytton. "Localization and quantification of endoplasmic reticulum Ca(2+)-ATPase isoform transcripts." American Journal of Physiology-Cell Physiology 269, no. 3 (September 1, 1995): C775—C784. http://dx.doi.org/10.1152/ajpcell.1995.269.3.c775.

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The Ca(2+)-adenosinetriphosphatase pump of the sarcoplasmic or endoplasmic reticulum (SERCA) plays a critical role in Ca2+ signaling and homeostasis in all cells and is encoded by a family of homologous and alternatively spliced genes. To understand more clearly the role the different isoforms play in cell physiology, we have undertaken a quantitative and qualitative assessment of the tissue distribution of transcripts encoding each SERCA isoform. SERCA1 expression is restricted to fast-twitch striated muscles, SERCA2a to cardiac and slow-twitch striated muscles, whereas SERCA2b is ubiquitously expressed. SERCA3 is expressed most abundantly in large and small intestine, thymus, and cerebellum and at lower levels in spleen, lymph node, and lung. In situ hybridization analyses revealed SERCA3 transcripts in cells of the intestinal crypt, the thymic cortex, and Purkinje cells in cerebellum. In addition, SERCA3 was expressed abundantly in isolated rat spleen lymphocytes, in various murine lymphoid cell lines, and in primary cultured microvascular endothelial cells. This analysis demonstrates that SERCA3 is expressed selectively in cells in which Ca2+ signaling plays a critical and sensitive role in regulating physiological processes.

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Helander, Anders, Gunne Eriksson, Helena Stibler, and Jan-Olof Jeppsson. "Interference of Transferrin Isoform Types with Carbohydrate-deficient Transferrin Quantification in the Identification of Alcohol Abuse." Clinical Chemistry 47, no. 7 (July 1, 2001): 1225–33. http://dx.doi.org/10.1093/clinchem/47.7.1225.

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Abstract Background: Isoforms of transferrin interfere with measurement of carbohydrate-deficient transferrin (CDT) as a marker of heavy alcohol consumption. We evaluated the rate of inaccurate CDT results by immunoassays. Methods: We studied 2360 consecutive sera (1614 individuals) submitted for CDT assay without clinical information as well as samples from 1 patient with a congenital disorder of glycosylation (CDG Ia) and from 6 healthy carriers of CDG Ia. The CDTect, %CDT-TIA, and new %CDT immunoassays were compared with HPLC (%CDT-HPLC). Transferrin isoform pattern were evaluated by isoelectric focusing (IEF). Results: Transferrin BC and CD heterozygotes were found at frequencies of ∼0.7% and ∼0.2%, respectively. Another transferrin C subtype, where di- and trisialotransferrin partly coeluted (tentatively identified as C2C3), was observed in ∼0.6%. Compared with the %CDT-HPLC method, the immunoassays often produced low results for transferrin BC and high results for transferrin CD and “C2C3”. A very high trisialotransferrin value (frequency ∼1%) often produced high CDT immunoassay results. In four of six healthy carriers of CDG Ia, a- and disialotransferrin were highly increased and the HPLC and IEF isoform patterns were indistinguishable from those in alcohol abuse. Conclusions: Rare transferrin isoform types and abnormal amounts of trisialotransferrin (total frequency ∼2–3%) may cause incorrect determination of CDT with immunoassays. The observed variants were readily identified by HPLC and IEF, which can be recommended for verification of CDT immunoassay results in doubtful cases. In healthy carriers of CDG Ia, CDT is high by all assays.

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Wu, K. D., and J. Lytton. "Molecular cloning and quantification of sarcoplasmic reticulum Ca(2+)-ATPase isoforms in rat muscles." American Journal of Physiology-Cell Physiology 264, no. 2 (February 1, 1993): C333—C341. http://dx.doi.org/10.1152/ajpcell.1993.264.2.c333.

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A cDNA encoding the full-length adult rat fast-twitch muscle Ca(2+)-adenosinetriphosphatase (ATPase) was cloned. The deduced amino acid sequence of this molecule has 97 and 90% identity with those of rabbit fast-twitch muscle and chicken skeletal muscle Ca(2+)-ATPases, respectively. Specific probes from the 3'-untranslated region of each sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPase (SERCA) gene product and full-length cRNA transcript standards were used to determine the quantity of mRNA encoding each isoform in various rat muscles. Quantitative immunoblotting was also used to determine the protein content of each SERCA isoform. Fast-twitch fibers expressed both SERCA1 mRNA and protein at a level two- to fivefold higher than SERCA2 was expressed in slow-twitch fibers. We observed a protein-to-mRNA ratio that varied from approximately 500,000 molecules per molecule in the fast-twitch muscles to approximately 200,000 in cardiac and smooth muscles. There was no difference, however, between the ratio for different isoforms in the same muscle. The content of Ca2+ pump in a given muscle therefore depends on at least three factors: 1) the efficiency of gene transcription and message stability (fiber type dependent), 2) the efficiency of translation and protein stability (muscle identity dependent), and 3) fiber composition of the muscle.

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Nowicka, Malgorzata, and Mark D. Robinson. "DRIMSeq: a Dirichlet-multinomial framework for multivariate count outcomes in genomics." F1000Research 5 (December 6, 2016): 1356. http://dx.doi.org/10.12688/f1000research.8900.2.

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There are many instances in genomics data analyses where measurements are made on a multivariate response. For example, alternative splicing can lead to multiple expressed isoforms from the same primary transcript. There are situations where differences (e.g. between normal and disease state) in the relative ratio of expressed isoforms may have significant phenotypic consequences or lead to prognostic capabilities. Similarly, knowledge of single nucleotide polymorphisms (SNPs) that affect splicing, so-called splicing quantitative trait loci (sQTL) will help to characterize the effects of genetic variation on gene expression. RNA sequencing (RNA-seq) has provided an attractive toolbox to carefully unravel alternative splicing outcomes and recently, fast and accurate methods for transcript quantification have become available. We propose a statistical framework based on the Dirichlet-multinomial distribution that can discover changes in isoform usage between conditions and SNPs that affect relative expression of transcripts using these quantifications. The Dirichlet-multinomial model naturally accounts for the differential gene expression without losing information about overall gene abundance and by joint modeling of isoform expression, it has the capability to account for their correlated nature. The main challenge in this approach is to get robust estimates of model parameters with limited numbers of replicates. We approach this by sharing information and show that our method improves on existing approaches in terms of standard statistical performance metrics. The framework is applicable to other multivariate scenarios, such as Poly-A-seq or where beta-binomial models have been applied (e.g., differential DNA methylation). Our method is available as a Bioconductor R package called DRIMSeq.

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Du, Jiang, Jing Leng, Lukas Habegger, Andrea Sboner, Drew McDermott, and Mark Gerstein. "IQSeq: Integrated Isoform Quantification Analysis Based on Next-Generation Sequencing." PLoS ONE 7, no. 1 (January 6, 2012): e29175. http://dx.doi.org/10.1371/journal.pone.0029175.

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Enk, Viktoria M., Christian Baumann, Michaela Thoß, Kenneth C. Luzynski, Ebrahim Razzazi-Fazeli, and Dustin J. Penn. "Regulation of highly homologous major urinary proteins in house mice quantified with label-free proteomic methods." Molecular BioSystems 12, no. 10 (2016): 3005–16. http://dx.doi.org/10.1039/c6mb00278a.

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Nowicka, Malgorzata, and Mark D. Robinson. "DRIMSeq: a Dirichlet-multinomial framework for multivariate count outcomes in genomics." F1000Research 5 (June 13, 2016): 1356. http://dx.doi.org/10.12688/f1000research.8900.1.

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There are many instances in genomics data analyses where measurements are made on a multivariate response. For example, alternative splicing can lead to multiple expressed isoforms from the same primary transcript. There are situations where the total abundance of gene expression does not change (e.g. between normal and disease state), but differences in the relative ratio of expressed isoforms may have significant phenotypic consequences or lead to prognostic capabilities. Similarly, knowledge of single nucleotide polymorphisms (SNPs) that affect splicing, so-called splicing quantitative trait loci (sQTL), will help to characterize the effects of genetic variation on gene expression. RNA sequencing (RNA-seq) has provided an attractive toolbox to carefully unravel alternative splicing outcomes and recently, fast and accurate methods for transcript quantification have become available. We propose a statistical framework based on the Dirichlet-multinomial distribution that can discover changes in isoform usage between conditions and SNPs that affect splicing outcome using these quantifications. The Dirichlet-multinomial model naturally accounts for the differential gene expression without losing information about overall gene abundance and by joint modeling of isoform expression, it has the capability to account for their correlated nature. The main challenge in this approach is to get robust estimates of model parameters with limited numbers of replicates. We approach this by sharing information and show that our method improves on existing approaches in terms of standard statistical performance metrics. The framework is applicable to other multivariate scenarios, such as Poly-A-seq or where beta-binomial models have been applied (e.g., differential DNA methylation). Our method is available as a Bioconductor R package called DRIMSeq.

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Liu, Junfeng, Ziyang An, Jianjun Luo, Jing Li, Feifei Li, and Zhihua Zhang. "Episo: quantitative estimation of RNA 5-methylcytosine at isoform level by high-throughput sequencing of RNA treated with bisulfite." Bioinformatics 36, no. 7 (December 3, 2019): 2033–39. http://dx.doi.org/10.1093/bioinformatics/btz900.

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Abstract Motivation RNA 5-methylcytosine (m5C) is a type of post-transcriptional modification that may be involved in numerous biological processes and tumorigenesis. RNA m5C can be profiled at single-nucleotide resolution by high-throughput sequencing of RNA treated with bisulfite (RNA-BisSeq). However, the exploration of transcriptome-wide profile and potential function of m5C in splicing remains to be elucidated due to lack of isoform level m5C quantification tool. Results We developed a computational package to quantify Epitranscriptomal RNA m5C at the transcript isoform level (named Episo). Episo consists of three tools: mapper, quant and Bisulfitefq, for mapping, quantifying and simulating RNA-BisSeq data, respectively. The high accuracy of Episo was validated using an improved m5C-specific methylated RNA immunoprecipitation (meRIP) protocol, as well as a set of in silico experiments. By applying Episo to public human and mouse RNA-BisSeq data, we found that the RNA m5C is not evenly distributed among the transcript isoforms, implying the m5C may subject to be regulated at isoform level. Availability and implementation Episo is released under the GNU GPLv3+ license. The resource code Episo is freely accessible from https://github.com/liujunfengtop/Episo (with Tophat/cufflink) and https://github.com/liujunfengtop/Episo/tree/master/Episo_Kallisto (with Kallisto). Supplementary information Supplementary data are available at Bioinformatics online.

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Jeffries, Elizabeth P., William I. Denq, Jonathan C. Bartko, and Michael A. Trakselis. "Identification, quantification, and evolutionary analysis of a novel isoform of MCM9." Gene 519, no. 1 (April 2013): 41–49. http://dx.doi.org/10.1016/j.gene.2013.01.054.

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Xu, Di, Giulianna R. Borges, Deborah R. Davis, Khristofor Agassandian, Maria Luisa S. Sequeira Lopez, R. Ariel Gomez, Martin D. Cassell, Justin L. Grobe, and Curt D. Sigmund. "Neuron- or glial-specific ablation of secreted renin does not affect renal renin, baseline arterial pressure, or metabolism." Physiological Genomics 43, no. 6 (March 2011): 286–94. http://dx.doi.org/10.1152/physiolgenomics.00208.2010.

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The renin-angiotensin system (RAS), known for its roles in cardiovascular, metabolic, and developmental regulation, is present in both the circulation and in many individual tissues throughout the body. Substantial evidence supports the existence of a brain RAS, though quantification and localization of brain renin have been hampered by its low expression levels. We and others have previously determined that there are two isoforms of renin expressed in the brain. The classical isoform encoding secreted renin (sREN) and a novel isoform encoding intracellular renin (icREN), the product of an alternative promoter and first exon (exon 1b). The differential role that these two isoforms play in cardiovascular and metabolic regulation remains unclear. Here we examined the physiological consequences of neuron- and glia-specific knockouts of sREN by crossing mice in which the sREN promoter and isoform-specific first exon (exon-1a) is flanked by LoxP sequences (sRENflox mice) with mice expressing Cre-recombinase controlled by either the neuron-specific Nestin promoter or the glia-specific GFAP promoter. Resulting offspring exhibited selective knockout of sREN in either neurons or glia, while preserving expression of icREN. Consistent with a hypothesized role of icREN in the brain RAS, neuron- and glia-specific knockout of sREN had no effect on blood pressure or heart rate; food, water, or sodium intake; renal function; or metabolic rate. These data demonstrate that sREN is dispensable within the brain for normal physiological regulation of cardiovascular, hydromineral, and metabolic regulation, and thereby indirectly support the importance of icREN in brain RAS function.

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Beach, C. M., M. C. De Beer, J. D. Sipe, L. D. Loose, and F. C. De Beer. "Human serum amyloid A protein. Complete amino acid sequence of a new variant." Biochemical Journal 282, no. 2 (March 1, 1992): 615–20. http://dx.doi.org/10.1042/bj2820615.

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Serum amyloid A protein (SAA), an acute-phase reactant and apolipoprotein of high-density lipoprotein, is a polymorphic protein with six reported isoforms. These are the products of three genes, i.e., cDNA pA1, cDNA pSAA82 and genomic DNA SAAg9, the last two being allelic variants at a single locus. We have identified an individual with additional novel SAA isoforms on isoelectric-focusing analysis. By using 3-bromo-3-methyl-2-(2′-nitrophenylsulphenyl)-indolenine (BNPS-skatole) cleavage of the protein at tryptophan residues we obtained the complete amino acid sequence of a novel isoform. Additional cleavage by endoproteinase Asp-N allowed verification of the tryptophan residues and complete amino acid sequence of both isoforms. The suitability of this approach to the rapid sequencing of SAA was demonstrated. Sequence analysis and quantification suggest that these isoforms are the result of the first confirmed allelic variation at the SAA1 locus. We designate the protein products of this allele SAA1 beta (pI 6.1) and SAA1 beta des-Arg (pI 5.6).

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Woldemariam, Getachew A., and Anthony W. Butch. "Immunoextraction–Tandem Mass Spectrometry Method for Measuring Intact Human Chorionic Gonadotropin, Free β-Subunit, and β-Subunit Core Fragment in Urine." Clinical Chemistry 60, no. 8 (August 1, 2014): 1089–97. http://dx.doi.org/10.1373/clinchem.2014.222703.

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Abstract BACKGROUND Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles. Because of the potential for abuse, hCG is banned (males only) in most sports and has been placed on the World Anti-Doping Agency list of prohibited substances. Intact hCG, free β-subunit (hCGβ), and β-subunit core fragment (hCGβcf) are the major variants or isoforms in urine. Immunoassays are used by antidoping laboratories to measure urinary hCG. Cross-reactivity with isoforms differs among immunoassays, resulting in widely varying results. We developed a sequential immunoextraction method with LC-MS/MS detection for quantification of intact hCG, hCGβ, and hCGβcf in urine. METHODS hCG isoforms were immunoextracted with antibody-conjugated magnetic beads and digested with trypsin, and hCGβ and hCGβcf unique peptides were quantified by LC-MS/MS with the corresponding heavy peptides as internal standard. hCG isoform concentrations were determined in urine after administration of hCG, and the intact hCG results were compared to immunoassay results. RESULTS The method was linear to 20 IU/L. Total imprecision was 6.6%–13.7% (CV), recovery ranged from 91% to 109%, and the limit of quantification was 0.2 IU/L. Intact hCG predominated in the urine after administration of 2 hCG formulations. The window of detection ranged from 6 to 9 days. Mean immunoassay results were 12.4–15.5 IU/L higher than LC-MS/MS results. CONCLUSIONS The performance characteristics of the method are acceptable for measuring hCG isoforms, and the method can quantify intact hCG and hCGβ separately. The limit of quantification will allow LC-MS/MS hCG reference intervals to be established in nondoping male athletes for improved doping control.

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CARGINALE, Vincenzo, Rosaria SCUDIERO, Clemente CAPASSO, Antonio CAPASSO, Peter KILLE, Guido di PRISCO, and Elio PARISI. "Cadmium-induced differential accumulation of metallothionein isoforms in the Antarctic icefish, which exhibits no basal metallothionein protein but high endogenous mRNA levels." Biochemical Journal 332, no. 2 (June 1, 1998): 475–81. http://dx.doi.org/10.1042/bj3320475.

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Reverse transcriptase-mediated PCR has been used to isolate two distinct metallothionein (MT) cDNA species from RNA extracted from icefish liver, namely MT-I and MT-II. Northern blot analysis with these cDNA species revealed that significant endogenous levels of MT mRNA were present in liver tissues of normal animals despite the fact that no MT protein could be found accumulating in the same tissue. However, multiple injections of CdCl2 induced high levels of both MT mRNA and MT protein. Sequence analysis of the cDNA species that were present after cadmium injection revealed the presence of both isoforms. Quantification of the MT-I and MT-II transcripts from normal and heavy-metal-treated fish showed an alteration in the ratio of the MT isoform transcripts. Endogenous transcripts consisted mostly of MT-II, whereas the MT-I transcript was preferentially accumulated only in response to the cadmium salt. The protein encoded by each cDNA isoform was isolated from the heavy-metal-treated fish and the availability of the specific MT mRNA for translation was demonstrated by translation in vitro. These results show that: (1) there is a discrepancy between the significant endogenous levels of MT mRNA and the absence of MT protein; (2) the accumulation of MT in icefish liver can be triggered by heavy metals; (3) genes encoding distinct MT isoforms are differentially regulated by heavy metals.

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Jin, Hyun Yong, Yanyan Tudor, Kaylee Choi, Zhifei Shao, Brian A. Sparling, Joseph G. McGivern, and Antony Symons. "High-Throughput Implementation of the NanoBRET Target Engagement Intracellular Kinase Assay to Reveal Differential Compound Engagement by SIK2/3 Isoforms." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 2 (December 18, 2019): 215–22. http://dx.doi.org/10.1177/2472555219893277.

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The real-time quantification of target engagement (TE) by small-molecule ligands in living cells remains technically challenging. Systematic quantification of such interactions in a high-throughput setting holds promise for identification of target-specific, potent small molecules within a pathophysiological and biologically relevant cellular context. The salt-inducible kinases (SIKs) belong to a subfamily of the AMP-activated protein kinase (AMPK) family and are composed of three isoforms in humans (SIK1, SIK2, and SIK3). They modulate the production of pro- and anti-inflammatory cytokines in immune cells. Although pan-SIK inhibitors are sufficient to reverse SIK-dependent inflammatory responses, the apparent toxicity associated with SIK3 inhibition suggests that isoform-specific inhibition is required to realize therapeutic benefit with acceptable safety margins. Here, we used the NanoBRET TE intracellular kinase assay, a sensitive energy transfer technique, to directly measure molecular proximity and quantify TE in HEK293T cells overexpressing SIK2 or SIK3. Our 384-well high-throughput screening of 530 compounds demonstrates that the NanoBRET TE intracellular kinase assay was sensitive and robust enough to reveal differential engagement of candidate compounds with the two SIK isoforms and further highlights the feasibility of high-throughput implementation of NanoBRET TE intracellular kinase assays for target-driven small-molecule screening.

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Zhang, Wei, Jae-Woong Chang, Lilong Lin, Kay Minn, Baolin Wu, Jeremy Chien, Jeongsik Yong, Hui Zheng, and Rui Kuang. "Network-Based Isoform Quantification with RNA-Seq Data for Cancer Transcriptome Analysis." PLOS Computational Biology 11, no. 12 (December 23, 2015): e1004465. http://dx.doi.org/10.1371/journal.pcbi.1004465.

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Schriebl, Kornelia, Evelyn Trummer, Robert Weik, Christine Lattenmayer, Dethardt Müller, Renate Kunert, Hermann Katinger, and Karola Vorauer-Uhl. "Applicability of different fluorescent dyes for isoform quantification on linear IPG gels." ELECTROPHORESIS 28, no. 12 (June 2007): 2100–2107. http://dx.doi.org/10.1002/elps.200600695.

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Li, Wei Vivian, Anqi Zhao, Shihua Zhang, and Jingyi Jessica Li. "MSIQ: Joint modeling of multiple RNA-seq samples for accurate isoform quantification." Annals of Applied Statistics 12, no. 1 (March 2018): 510–39. http://dx.doi.org/10.1214/17-aoas1100.

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Zhang, Jing, C. C. Jay Kuo, and Liang Chen. "WemIQ: an accurate and robust isoform quantification method for RNA-seq data." Bioinformatics 31, no. 6 (November 28, 2014): 878–85. http://dx.doi.org/10.1093/bioinformatics/btu757.

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Henderson, Clark M., Pamela L. Lutsey, Jeffrey R. Misialek, Thomas J. Laha, Elizabeth Selvin, John H. Eckfeldt, and Andrew N. Hoofnagle. "Measurement by a Novel LC-MS/MS Methodology Reveals Similar Serum Concentrations of Vitamin D–Binding Protein in Blacks and Whites." Clinical Chemistry 62, no. 1 (January 1, 2016): 179–87. http://dx.doi.org/10.1373/clinchem.2015.244541.

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Abstract BACKGROUND Vitamin D deficiency is associated with poor bone health and other adverse health outcomes; however, the associations are greatly attenuated in black vs white individuals. One possible explanation for this attenuation is different concentrations of bioavailable vitamin D metabolites in plasma, which are estimated with equations that include the total concentration of vitamin D binding globulin (VDBG) and haplotype-specific dissociation constants. METHODS We developed a method to quantify VDBG with LC-MS/MS that could also identify the haplotypes/isoforms of VDBG present. We validated the method according to recent recommendations for publications of biomarker studies. We determined serum VDBG concentrations in samples from the Atherosclerosis Risk in Communities cohort and compared the results with a widely used monoclonal immunoassay. RESULTS With 10 μL of serum or plasma, the lower limit of quantification for the assay (<20% CV) was 71 μg/mL. The assay was linear from 62 to 434 μg/mL, with total imprecision of 7.3–9.0% CV at approximately 250 μg/mL. Significant hemolysis interfered with quantification. The identification of isoforms was 97% concordant with genotyping (κ coefficient). Method comparison with immunoassay revealed significant isoform-specific effects in the immunoassay. Mean concentrations (SD) of VDBG by mass spectrometry were similar in whites and blacks [262 (25) vs 266 (35) μg/mL, respectively; P = 0.43]. CONCLUSIONS Validated mass spectrometric methods for the quantification of proteins in human samples can provide additional information beyond immunoassay. Counter to prior observations by immunoassay, VDBG concentrations did not vary by race.

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Levin, Michal, Harel Zalts, Natalia Mostov, Tamar Hashimshony, and Itai Yanai. "Gene expression dynamics are a proxy for selective pressures on alternatively polyadenylated isoforms." Nucleic Acids Research 48, no. 11 (May 18, 2020): 5926–38. http://dx.doi.org/10.1093/nar/gkaa359.

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Abstract Alternative polyadenylation (APA) produces isoforms with distinct 3′-ends, yet their functional differences remain largely unknown. Here, we introduce the APA-seq method to detect the expression levels of APA isoforms from 3′-end RNA-Seq data by exploiting both paired-end reads for gene isoform identification and quantification. We detected the expression levels of APA isoforms in individual Caenorhabditis elegans embryos at different stages throughout embryogenesis. Examining the correlation between the temporal profiles of isoforms led us to distinguish two classes of genes: those with highly correlated isoforms (HCI) and those with lowly correlated isoforms (LCI) across time. We hypothesized that variants with similar expression profiles may be the product of biological noise, while the LCI variants may be under tighter selection and consequently their distinct 3′ UTR isoforms are more likely to have functional consequences. Supporting this notion, we found that LCI genes have significantly more miRNA binding sites, more correlated expression profiles with those of their targeting miRNAs and a relative lack of correspondence between their transcription and protein abundances. Collectively, our results suggest that a lack of coherence among the regulation of 3′ UTR isoforms is a proxy for selective pressures acting upon APA usage and consequently for their functional relevance.

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Patro, Rob, Stephen M. Mount, and Carl Kingsford. "Sailfish enables alignment-free isoform quantification from RNA-seq reads using lightweight algorithms." Nature Biotechnology 32, no. 5 (April 20, 2014): 462–64. http://dx.doi.org/10.1038/nbt.2862.

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Deng, Yue, Feng Bao, Yang Yang, Xiangyang Ji, Mulong Du, Zhengdong Zhang, Meilin Wang, and Qionghai Dai. "Information transduction capacity reduces the uncertainties in annotation-free isoform discovery and quantification." Nucleic Acids Research 45, no. 15 (July 7, 2017): e143-e143. http://dx.doi.org/10.1093/nar/gkx585.

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Bernard, Elsa, Laurent Jacob, Julien Mairal, and Jean-Philippe Vert. "Efficient RNA isoform identification and quantification from RNA-Seq data with network flows." Bioinformatics 30, no. 17 (May 9, 2014): 2447–55. http://dx.doi.org/10.1093/bioinformatics/btu317.

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Lindqvist, Arne, Helena Källström, Andreas Lundgren, Emad Barsoum, and Christina Karlsson Rosenthal. "Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1–Cdk1 at the centrosome." Journal of Cell Biology 171, no. 1 (October 10, 2005): 35–45. http://dx.doi.org/10.1083/jcb.200503066.

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Cdc25 phosphatases are essential for the activation of mitotic cyclin–Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1–Cdk1 and cyclin A–Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1–Cdk1 on centrosomes.

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Parsons, J. Kellogg, Elizabeth A. Saria, Masashi Nakayama, Robert L. Vessella, Charles L. Sawyers, William B. Isaacs, Dennis A. Faith, et al. "Comprehensive mutational analysis and mRNA isoform quantification ofTP63in normal and neoplastic human prostate cells." Prostate 69, no. 5 (April 1, 2009): 559–69. http://dx.doi.org/10.1002/pros.20904.

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Bellani, Marina A., Kingsley A. Boateng, Dianne McLeod, and R. Daniel Camerini-Otero. "The Expression Profile of the Major Mouse SPO11 Isoforms Indicates that SPO11β Introduces Double Strand Breaks and Suggests that SPO11α Has an Additional Role in Prophase in both Spermatocytes and Oocytes." Molecular and Cellular Biology 30, no. 18 (July 20, 2010): 4391–403. http://dx.doi.org/10.1128/mcb.00002-10.

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ABSTRACT Both in mice and humans, two major SPO11 isoforms are generated by alternative splicing: SPO11α (exon 2 skipped) and SPO11β. Thus, the alternative splicing event must have emerged before the mouse and human lineages diverged and was maintained during 90 million years of evolution, arguing for an essential role for both isoforms. Here we demonstrate that developmental regulation of alternative splicing at the Spo11 locus governs the sequential expression of SPO11 isoforms in male meiotic prophase. Protein quantification in juvenile mice and in prophase mutants indicates that early spermatocytes synthesize primarily SPO11β. Estimation of the number of SPO11 dimers (ββ/αβ/αα) in mutants in which spermatocytes undergo a normal number of double strand breaks but arrest in midprophase due to inefficient repair argues for a role for SPO11β-containing dimers in introducing the breaks in leptonema. Expression kinetics in males suggested a role for SPO11α in pachytene/diplotene spermatocytes. Nevertheless, we found that both alternative transcripts can be detected in oocytes throughout prophase I, arguing against a male-specific function for this isoform. Altogether, our data support a role for SPO11α in mid- to late prophase, presumably acting as a topoisomerase, that would be conserved in male and female meiocytes.

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Apple, F. S., Y. Hellsten, and P. M. Clarkson. "Early detection of skeletal muscle injury by assay of creatine kinase MM isoforms in serum after acute exercise." Clinical Chemistry 34, no. 6 (June 1, 1988): 1102–4. http://dx.doi.org/10.1093/clinchem/34.6.1102.

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Abstract We could detect skeletal muscle injury early after an acute exercise bout by measuring creatine kinase (CK, EC 2.7.3.2) MM isoforms in serum. Eleven men performed 120 alternating-arm, eccentric (muscle lengthening) biceps contractions with the intensity of each contraction being 110% of maximal concentric strength--a form of exercise previously shown to cause significant increases of CK in serum at 24 h and muscle soreness 48 h after exercise. Total CK and CK-MM isoform activities in serum were determined before and at 0.5, 0.75, 1, 1.5, 2, and 6 h after exercise. Using thin-film agarose gels and a rapid isoelectric focusing technique, we separated the MM isoforms into MM3 (skeletal muscle form), MM2, and MM1 (in vivo conversion forms). The isoforms reflected the MM form released into the serum from tissue as well as the conversion of one form to another. There were no significant increases in total CK from before to 6 h after exercise: 75 (SD 36) vs 91 (SD 33) U/L. However, CK MM3 in serum increased significantly (P less than 0.01) within 2 h after exercise from 22 (SD 6)% to 28 (SD 6)%. The MM3 to MM1 ratio also increased significantly (P less than 0.05) during this time, from 0.6 (SD 0.3) to 0.9 (SD 0.4). Thus, quantification of CK MM isoforms permitted very early detection of skeletal muscle enzyme release.

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Farquharson, C., A. S. Law, E. Seawright, D. W. Burt, and C. C. Whitehead. "The expression of transforming growth factor-β by cultured chick growth plate chondrocytes: differential regulation by 1,25-dihydroxyvitamin D3." Journal of Endocrinology 149, no. 2 (May 1996): 277–85. http://dx.doi.org/10.1677/joe.0.1490277.

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Abstract 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and transforming growth factor-β (TGF-β) are both important regulators of chondrocyte growth and differentiation. We report here that 1,25(OH)2D3 differentially regulates the expression of the genes for TGF-β1 to -β3 and the secretion of the corresponding proteins in cultured chick chondrocytes. Confluent growth plate chondrocytes were serum-deprived and cultured in varying concentrations of 1,25(OH)2D3. Cells were assayed for TGF-β mRNA and conditioned medium was assayed for TGF-β activity and isoform composition. Active TGF-β was only detected in 10−8m 1,25(OH)2D3-treated cultures (8·37 ng active TGF-β/mg protein). There was a significant decrease in total (latent+active) TGF-β activity in conditioned medium of 10−12 m (23·4%; P<0·05) and 10−10 m (20·7%; P<0·05) 1,25(OH)2D3-treated cultures but 10−8 m 1,25(OH)2D3 significantly increased (30·9%; P<0·01) TGF-β activity. The amounts of TGF-β1, -β2 and -β3 isoforms produced were similar in control, 10−10 or 10−12m 1,25(OH)2D3-treated cultures but the conditioned medium of 10−8 m 1,25(OH)2D3-treated cultures contained significantly higher amounts of all three isoforms. Quantification of TGF-β mRNA demonstrated differential control of TGF-β gene expression with TGF-β1 and -β3 mRNA levels reduced by all concentrations of 1,25(OH)2D3 examined (10−8, 10−10 and 10−12 m) whilst TGF-β2 mRNA concentrations were elevated. Our results indicated that 1,25(OH)2D3 regulates chick growth plate chondrocyte TGF-β secretion and mRNA expression in a concentration-dependent and isoform-specific manner. This interaction may be important in the regulation of chondrocyte metabolism and endochondral bone growth. Journal of Endocrinology (1996) 149, 277–285

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Depreux, F. F. S., C. S. Okamura, D. R. Swartz, A. L. Grant, A. M. Brandstetter, and D. E. Gerrard. "Quantification of myosin heavy chain isoform in porcine muscle using an enzyme-linked immunosorbent assay." Meat Science 56, no. 3 (November 2000): 261–69. http://dx.doi.org/10.1016/s0309-1740(00)00051-6.

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Mourah, Samia, Raphaël Porcher, Géraldine Lescaille, Philippe Rousselot, Marie-Pierre Podgorniak, Géraldine Labarchède, Benyoussef Naïmi, Jacques Medioni, Hervé Dombret, and Fabien Calvo. "Quantification of VEGF Isoforms and VEGFR Transcripts by qRT-PCR and Their Significance in Acute Myeloid Leukemia." International Journal of Biological Markers 24, no. 1 (January 2009): 22–31. http://dx.doi.org/10.1177/172460080902400104.

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Vascular endothelial growth factor (VEGF) and its receptors are known to play an important role in normal and pathological hematopoiesis but the prognostic impact of VEGF isoform transcripts in acute myeloid leukemia (AML) has not been addressed. We conducted a single-institution prospective study to analyze the impact of these angiogenic factors and the expression of their receptors on the survival of adult patients newly diagnosed with AML. We investigated the levels of VEGF transcript isoforms VEGF121, -145, -165, -189 and -206 and their receptors, VEGFR-1 and VEGFR-2, using quantitative reverse transcriptase polymerase chain reaction assays in peripheral blood mononuclear cells (PBMCs) of 67 consecutive AML patients at diagnosis. VEGF total protein was measured for comparison with mRNA levels in PBMCs. The VEGF121 splice variant transcript in AML PBMCs was significantly higher than in the normal controls. VEGF transcripts were quantified in all samples while its protein was detected in 42/67 (63%) of AML samples. High levels of VEGF121, VEGF165 transcripts and VEGF protein in AML were significantly related to a worse prognosis when analyzing overall survival (p<0.0001, p=0.019 and p=0.012, respectively) or event-free survival (p<0.0001, p=0.010 and p=0.047) using univariate analysis. In multivariable analysis only VEGF121 expression remained an independent prognostic factor for either event-free survival or overall survival [aHR=8.83 (3.48–22.4), p<0.0001, and aHR=9.52 (3.41–26.6), p<0.0001]. No prognostic value was observed for the other isoforms and the two receptors. Our findings show that the level of VEGF121 mRNA in circulating cells from AML patients is a strong independent prognostic parameter, which could be useful in the management of unselected AML patients.

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Taddei-Peters, W. C., B. T. Butman, G. R. Jones, T. M. Venetta, P. F. Macomber, and J. H. Ransom. "Quantification of lipoprotein(a) particles containing various apolipoprotein(a) isoforms by a monoclonal anti-apo(a) capture antibody and a polyclonal anti-apolipoprotein B detection antibody sandwich enzyme immunoassay." Clinical Chemistry 39, no. 7 (July 1, 1993): 1382–89. http://dx.doi.org/10.1093/clinchem/39.7.1382.

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Abstract A quantitative sandwich ELISA for lipoprotein(a) [Lp(a)], utilizing a monoclonal capture antibody that recognizes human and rhesus monkey apolipoprotein(a) [apo(a)] isoforms in combination with a polyclonal anti-apolipoprotein B-peroxidase conjugate was developed. This assay generates a linear calibration curve from 31.2 to 1000 mg/L, is highly reproducible (intra- and interassay CV of < 5% and < or = 12%, respectively), and shows no interference from plasminogen (1 g/L), low-density lipoprotein (6.00 g/L), triglycerides (27.00 g/L from chylomicrons and 10.00 g/L from very-low-density lipoprotein), hemoglobin (5 g/L), or bilirubin (30 mg/L). This assay format quantifies the concentration of Lp(a) on an equal molar basis regardless of apo(a) isoform. In contrast, a commercially available ELISA [Macra Lp(a)] method with a monoclonal anti-apo(a) capture antibody and a polyclonal anti-apo(a) conjugate was found to underestimate the Lp(a) concentrations of individuals with lower-M(r) apo(a) isoforms--whether quantifying the Lp(a) in plasma or the purified lipoprotein. This demonstrates the importance of assay format selection in quantifying Lp(a).

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Soneson, Charlotte, Michael I. Love, and Mark D. Robinson. "Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences." F1000Research 4 (December 30, 2015): 1521. http://dx.doi.org/10.12688/f1000research.7563.1.

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High-throughput sequencing of cDNA (RNA-seq) is used extensively to characterize the transcriptome of cells. Many transcriptomic studies aim at comparing either abundance levels or the transcriptome composition between given conditions, and as a first step, the sequencing reads must be used as the basis for abundance quantification of transcriptomic features of interest, such as genes or transcripts. Several different quantification approaches have been proposed, ranging from simple counting of reads that overlap given genomic regions to more complex estimation of underlying transcript abundances. In this paper, we show that gene-level abundance estimates and statistical inference offer advantages over transcript-level analyses, in terms of performance and interpretability. We also illustrate that while the presence of differential isoform usage can lead to inflated false discovery rates in differential expression analyses on simple count matrices and transcript-level abundance estimates improve the performance in simulated data, the difference is relatively minor in several real data sets. Finally, we provide an R package (tximport) to help users integrate transcript-level abundance estimates from common quantification pipelines into count-based statistical inference engines.

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Rochat, Bertrand, Davide Peduzzi, Justin McMullen, Amélie Favre, Emmanuel Kottelat, Bernard Favrat, Jean-Daniel Tissot, Anne Angelillo-Scherrer, Maciej Bromirski, and Sophie Waldvogel. "Validation of hepcidin quantification in plasma using LC–HRMS and discovery of a new hepcidin isoform." Bioanalysis 5, no. 20 (October 2013): 2509–20. http://dx.doi.org/10.4155/bio.13.225.

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Trapnell, Cole, Brian A. Williams, Geo Pertea, Ali Mortazavi, Gordon Kwan, Marijke J. van Baren, Steven L. Salzberg, Barbara J. Wold, and Lior Pachter. "Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation." Nature Biotechnology 28, no. 5 (May 2010): 511–15. http://dx.doi.org/10.1038/nbt.1621.

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Hu, Yu, Yichuan Liu, Xianyun Mao, Cheng Jia, Jane F. Ferguson, Chenyi Xue, Muredach P. Reilly, Hongzhe Li, and Mingyao Li. "PennSeq: accurate isoform-specific gene expression quantification in RNA-Seq by modeling non-uniform read distribution." Nucleic Acids Research 42, no. 3 (December 20, 2013): e20-e20. http://dx.doi.org/10.1093/nar/gkt1304.

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Potau, Josep Maria, Rosa Artells, Carmen Muñoz, Júlia Arias-Martorell, Juan Francisco Pastor, Félix Jesús de Paz, Mercedes Barbosa, Gaëlle Bello-Hellegouarch, and Alejandro Pérez-Pérez. "Quantification of Myosin Heavy Chain Isoform mRNA Transcripts in the Supraspinatus Muscle of Vertical Clinger Primates." Folia Primatologica 88, no. 6 (2017): 497–506. http://dx.doi.org/10.1159/000485246.

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Lemon, Douglas D., Philip J. Papst, Kristin Joly, Craig F. Plato, and Timothy A. McKinsey. "A high-performance liquid chromatography assay for quantification of cardiac myosin heavy chain isoform protein expression." Analytical Biochemistry 408, no. 1 (January 2011): 132–35. http://dx.doi.org/10.1016/j.ab.2010.08.041.

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Journal articles on the topic 'Isoform quantification'

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