A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells.
An important aim in the post-sequencing age of functional genomics is to translate gene sequences into protein functions. This shift of focus is particularly necessary for a very large number of human genes, referred to as novel genes, where we have no or very rudimentary information about their biochemical functions. Recently, a new method for investigating human gene functions using small interfering RNA molecules (siRNAs) has become available. siRNAs are powerful reagents for post-transcriptional silencing, where mRNA targeted by the siRNAs is degraded in vivo and the level of the encoded protein is reduced. However, the lack of antibodies against proteins encoded by novel genes restricts the general value of siRNAs as functional tools, as only the mRNA levels can be measured for these genes. We report a method that combines measurements of protein levels in cell culture for novel, exogenously expressed genes, with parallel measurements of the endogenous mRNA levels of the same genes. We find that this combinatorial approach correctly predicts siRNAs that efficiently reduce mRNA and protein levels in cultured cells. Furthermore, this method identifies proteins that have a slow turnover, which weakens the value of the RNA interference method as a tool for functional studies of such genes. The described method should prove to be valuable for large-scale functional studies of novel human genes.
Wu
Weilin
W
Center for Genomics and Bioinformatics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Hodges
Emily
E
Redelius
Jenny
J
Höög
Christer
C
D015153:Blotting, Western; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018076:DNA, Complementary; D011506:Proteins; D034741:RNA, Small Interfering; D012313:RNA