LEM-PCR: a method for determining relative transcript isoform proportions using real-time PCR without a standard curve.
Many genes express multiple transcript isoforms generated by alternative splicing of mRNA. Using real-time PCR, it is straightforward to determine the relative expression level of each isoform independently. However, it is less trivial to determine the relative proportions of different isoforms in a cDNA sample. The relative proportions of different isoforms can be important, as a small change in a highly abundant transcript may be more relevant than a large change in a minimally expressed transcript. Currently, determining the relative proportions of isoforms requires the construction of a standard curve using recombinant plasmid DNA or genomic DNA. As recombinant or genomic DNA standards often amplify with different efficiencies to cDNA samples, they may give under- or overestimations of isoform abundances. The method described in this article uses a titration curve generated from the same cDNA samples measured in the experiment. By using samples with different levels of separate isoforms, it is possible to derive linear equations which, when solved, allow the determination of the proportion of each isoform within the samples under study.
Virtue
S
S
Institute of Metabolic Science, Metabolic Research Laboratories, University of Cambridge, Box 289, Level 4, Addenbrooke's Hospital, Cambridge, UK. sv234@medschl.cam.ac.uk
Dale
M
M
Sethi
J K
JK
Vidal-Puig
A
A
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism / Q000502:physiology; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D051379:Mice; D008954:Models, Biological; D047495:PPAR gamma / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D020033:Protein Isoforms / Q000032:analysis* / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards
D047495:PPAR gamma; D020033:Protein Isoforms; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S