Quantification of ribozyme target RNA using real-time PCR.
An important part of the ribozyme efficiency-screening process is to have a fast and accurate way to measure steady-state levels of the target RNA. Here, we describe the use of real-time polymerase chain reaction (PCR) for quantification of ribozyme target transcripts. In contrast to classical quantitative PCR, real-time PCR does not require extensive manipulation or generation of relatively complex reagents, thus reducing the risk of contamination. PCR products generated by Taq polymerase in the presence of SYBR Green dye I can be monitored each cycle by collecting fluorescence signals emitted only as the double-stranded DNA is formed. The temperature at which the fluorescent data used for quantification are collected is based on the melting-curve analysis of the amplified product. After constructing a standard curve by plotting the log of the standards' copy number vs their fractional cycle number, the copy number of the unknown samples is automatically determined by interpolation of this curve. However it is very important to validate the melting curve profile with standard gel electrophoresis, particularly while setting up the technique. Real-time PCR is fast and reproducible. Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 h. The coefficient of variance is 15% in the range of 104-106 gene copies.
Klein
Dagmar
D
Diabetes Research Institute, University of Miami School of Medicine, FL, USA.
Ricordi
Camillo
C
Pastori
Ricardo L
RL
D001483:Base Sequence; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D013816:Thermodynamics
D017931:DNA Primers; D016337:RNA, Catalytic; D012313:RNA