Quantification of tumor suppressor mRNA expression by poly-competitive RT-PCR using a TS-IS that contained multiple internal competitors.
Despite the recent introduction of real-time PCR methods and cDNA microarrays, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. In this study, we developed a construct, termed tumor suppressor-internal standard (TS-IS) that produced polycompetitive RNA templates as an internal standard to quantify cellular RNA concentration of tumor suppressor genes. This construct is composed of not only sets of primers for detecting the expression of several tumor suppressor genes (such as pRB, p16(INK4A) 15(INK4B), p14(ARF) p53, and p21(WAF1)), but also HPRT as an endogenous marker. Using an internal standard RNA that was synthesized from the TS-IS construct, we were able to establish optimized conditions for the quantification of tumor suppressor genes with minimal amounts (50 ng) of cellular RNA. In addition, the usefulness of this method was confirmed by analyzing the expression levels of tumor suppressor genes in fourteen hepatoma cell lines as a model. The TS-IS assay that we used was inexpensive and a widely applicable method that permitted the reliable and accurate quantification of tumor suppressor genes.
Jung
Hee-Jung
HJ
Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Korea.
Chae
Ji Hyung
JH
Kim
Hoguen
H
Park
Jeon Han
JH
Lee
Jong Soo
JS
Choi
Woong Hwan
WH
Kim
Chul Geun
CG
D006528:Carcinoma, Hepatocellular / Q000235:genetics; D017931:DNA Primers; D020869:Gene Expression Profiling; D006801:Humans; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D017931:DNA Primers; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins