I would like to know if it is a common reagent kit for the target amplification of both DNA and RNA, or does it show amplification only for the RNA targets. Will the presence of DNA in the sample affect the reaction and its results?
Hello Janani,
You cannot use the AgPath One-Step RT-PCR reagents kit for amplification of DNA targets. The reagents are designed for amplification of RNA targets using the real time reverse transcription PCR strategy. It contains the highly efficient reverse transcriptase, MMLV RT that gives high cDNA yield.
You may face a problem if your sample is contaminated with genomic DNA. Because of the sequence identity of genomic DNA and cDNA which is derived from mRNA, non-specific amplification can occur which will have an influence on the accuracy of the results. The presence of genomic DNA contamination is likely to lead to an overestimation of the abundance of target mRNA in gene expression analysis.
Generally, DNA targets are detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest.
Best.
YES, you can, just before starting you have to add a step of RT deactivation (e.g. 94°C for 10 min) at the beginning of your PCR thermoprofile. However, the cost of the run will be more expensive when using RT-mastermix than ordinary PCR-mastermix.
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