Quantitation of tropoelastin mRNA and assessment of alternative splicing in human skin fibroblasts by reverse transcriptase-polymerase chain reaction.
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.
Holzenberger
M
M
Department of Surgery, UMDNJ-Robert Wood Johnson Medical School, New Brunswick 08903.
Levi-Minzi
S A
SA
Herzog
C P
CP
Deak
S B
SB
Robert
L
L
Boyd
C D
CD
D017398:Alternative Splicing; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase; D012867:Skin / Q000378:metabolism*; D014333:Tropoelastin / Q000096:biosynthesis* / Q000235:genetics
D017931:DNA Primers; D012333:RNA, Messenger; D014333:Tropoelastin; D012194:RNA-Directed DNA Polymerase