DR.Dhafer A.F…..Drawback Real-Time qRT-PCR
Dhafer A.F Alkoofee Ph.D, Biochemistry and Molecular Biology Dept. of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Kufa ORCiD .org/0000-0003-0046-4422
Drawback Real-Time qRT-PCR
We can summarized some of common hazards of qRT-PCR in the that should be taken in consideration when we design an experiment:
1. Inferior Primer and Probe Design: We strongly recommend using reliable primer design software for most efficient PCR primer and probe sets. These software uses adjustable parameters for optimal primer and probe design.
2. Using bad quality of RNA: Degraded or impure RNA can limit the efficiency of the RT reaction and reduce yield. RNA should either be prepared from fresh tissue, or from tissue treated with an RNA stabilization solution.
3. Not using master mix: qRT-PCR is a highly sensitive tool for analyzing RNA. As the PCR amplifies the target, errors are simultaneously amplified. Therefore, variability should be kept to a minimum whenever possible. A master mix should be used when setting up multiple reactions to minimize sample-to-sample and well-to-well variation and improve reproducibility.
4. The efficiency of the reaction is outside the acceptable range: The efficiency of the PCR should be 90–110% ( 3.6 > slope > 3.1), A number of variables can affect the efficiency of the PCR. These factors can include length of the amplicon, secondary structure, and primer design. Although valid data can be obtained that fall outside of the efficiency range, the qRT-PCR should be further optimized or alternative amplicon designed.