Hello Everyone,
I wanted to run RT-PCR using onestep real-time PCR systems and SYBR green (ThermoFischer) to check the gene expression in bacteria. I wanted to directly use 50 ng of DNA samples extracted from bacteria. The Master-mix includes DNA template, DEPC water, forward and reverse primers. The reverse transcriptase enzyme was excluded since i'm not using RNA as a template. I wanted to know whether my protocol is wright?
Looking forward to all.
Thanks and regards,
Arun
Using qPCR on DNA sample would not yield any information on gene expression. you can only determine CNVs or SNPs but not expression.
Hi. Some times I use genomic DNA (human) specific primers and probes to check DNA contamination of cDNA samples. If you try to estimate gene expression with only DNA it seems not possible on the one hand. But on the other hand you can estimate epigenetisc that can provide you a lot of information about expression regulation. Ater that you can verify it on the model with cDNA.
Since you are using DNA as a template you should follow the conventional PCR. However, it would not help to estimate gene expression.
It is better to extract RNA and follow the gene expression study, but if you still want to use DNA as a template - you can sequence the PCR product to know about your target gene. Whether it is a constitutive gene or facultative gene and blasting the sequence might give an answer to your desire.
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