Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions.
The recent development of real-time PCR has offered the opportunity of sensitive and accurate quantification of mRNA levels that is crucial in biomedical research. Although reverse transcription (RT)-PCR is at present the most sensitive method available, many low abundant mRNAs are, although detectable, often not quantifiable. Here we report an improved two-step real-time RT-PCR procedure using SYBR green I and the LightCycler that better permits accurate quantification of mRNAs. Omission of dithiothreitol from the cDNA synthesis reaction was found to be crucial. This resulted in a lower cycle number at which the cDNA level is determined (C(T) value), steeper amplification curves, and removal of background fluorescence in the subsequent PCR. In addition, the choice of the cDNA priming oligo can improve detection sensitivity even further. In contrast to hexamer primer usage, both gene-specific and oligo-dT(VN) priming were very efficient and accurate, with gene-specific priming being the most sensitive. Finally, accurate quantification of mRNAs by real-time PCR using SYBR green I requires verification of the specificity of PCR by both melting curve and gel analysis.
Experimental and Molecular Cardiology Group, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. r.h.lekanne@amc.uva.nl
Ronald H
RH
Lekanne Deprez
Arnoud C
AC
Fijnvandraat
Jan M
JM
Ruijter
Antoon F M
AF
Moorman
D000818:Animals; D052160:Benzothiazoles; D004247:DNA / Q000032:analysis; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis*; D003959:Diamines; D005456:Fluorescent Dyes; D015636:Magnesium Chloride / Q000494:pharmacology; D051379:Mice; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature
D052160:Benzothiazoles; D017931:DNA Primers; D018076:DNA, Complementary; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; D015636:Magnesium Chloride; C098022:SYBR Green I; D004247:DNA