Quantitation of tropoelastin mRNA and assessment of alternative splicing in human skin fibroblasts by reverse transcriptase-polymerase chain reaction.
Open Access
- 1 October 1993
- journal article
- Published by Cold Spring Harbor Laboratory in Genome Research
- Vol. 3 (2) , 107-114
- https://doi.org/10.1101/gr.3.2.107
Abstract
We have developed a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the quantitative measurement of levels of tropoelastin mRNA in total RNA preparations from skin fibroblasts. This method facilitates the reproducible detection of low abundance tropoelastin mRNA in the range of 10-1000 copies per cell. The procedure is based on a competitive RT-PCR assay where a tropoelastin cDNA-derived internal RNA standard is cotranscribed and coamplified together with the sample derived-endogenous target mRNA. In addition, RT-PCR of several domains of tropoelastin mRNA, followed by DNA sequence analysis of asymmetric PCR products, revealed a previously unknown pattern of alternate exon usage at the 3' end of the tropoelastin gene in human skin fibroblasts.Keywords
This publication has 37 references indexed in Scilit:
- A Restriction Fragment Length Polymorphism Results in a Nonconservative Amino Acid Substitution Encoded within the First Exon of the Human Lysyl Oxidase GeneGenomics, 1993
- Quantitative determination of rare mRNA species by PCR and solid-phase minisequencing.Genome Research, 1992
- Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloningBiochemistry, 1990
- Heterogeneity of Elastin Expression in Cutis Laxa Fibroblast StrainsJournal of Investigative Dermatology, 1989
- Structure of the bovine elastin gene and S1 nuclease analysis of alternative splicing of elastin mRNA in the bovine nuchal ligamentBiochemistry, 1989
- Cloning of Full-length Elastin cDNAs from a Human Skin Fibroblast Recombinant cDNA Library: Further Elucidation of Alternative Splicing Utilizing Exon-specific OligonucleotidesJournal of Investigative Dermatology, 1988
- Increased Elastin Production by Progeria Skin Fibroblasts is Controlled by the Steady-State Levels of Elastin mRNAJournal of Investigative Dermatology, 1988
- Repeating structure of chick tropoelastin revealed by complementary DNA cloningBiochemistry, 1987
- Structure of the 3’ Region of the Human Elastin Gene: Great Abundance of Alu Repetitive Sequences and Few Coding SequencesConnective Tissue Research, 1987
- Developmental changes in collagen and elastin biosynthesis in the porcine aortaDevelopmental Biology, 1986