Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR.
Real-time PCR is a novel technology recently described to perform quantitative analysis of amplified products. Unlike classical quantitative PCR, this method is easy to standardize, does not required extensive manipulation, and is not reagent intensive, so that the risk of contamination is minimized. Therefore, we have chosen reverse transcriptase real-time PCR to quantitate CD95 (Fas) transcripts to test the cleavage efficiency of anti-Fas ribozymes in the mouse insulinoma cell line beta TC-3. Based on the melting-curve analysis of the amplified products, we determined the temperature at which to collect the fluorescent data used for quantification. After constructing a standard curve by plotting the log of the standards' copy number versus their fractional cycle number, the copy numbers of the unknown samples were automatically determined by interpolation of this curve. As we illustrate in this study, it is important, particularly while setting up the technique, to validate the melting-curve profile with standard gel electrophoresis analysis, achieved by matching melting temperature and size of the amplified product. The method is fast and reproducible: Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 hr. The coefficient of variance is 15% in the range of 10(4)-10(6) gene copies. Accordingly, reverse transcriptase (RT) real-time PCR is a technique suitable for screening a large number of ribozymes.
Klein
D
D
Diabetes Research Institute, University of Miami School of Medicine, FL 33136, USA.
Denis
M
M
Ricordi
C
C
Pastori
R L
RL
D000818:Animals; D015152:Blotting, Northern; D016207:Cytokines / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005456:Fluorescent Dyes; D007340:Insulinoma / Q000378:metabolism; D051379:Mice; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000235:genetics*
D016207:Cytokines; D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D016337:RNA, Catalytic; D012333:RNA, Messenger; D019014:fas Receptor