I'm doing a RT-PCR on orchids, but the program I use now takes up to 3 hours. It would be nice if I could reduce that time with Phire. However, I'm not sure if it affects the gene-expression. Are the differences in expression still visible?
It should be fine, although make sure you do your RT step beforehand as the one I found online doesn't have an RT enzyme in the enzyme mix. Otherwise if you use the same enzyme as you do for your controls the relative or absolute expression levels will be the same. You may have different Ct values vs. a different enzyme but once you analyze the data the ddCt or absolute expression level should be the same or close to it.
For RT-PCR you need reverse transcriptase enzyme that will convert your RNA to cDNA. As far as i know Phire Hot Start II DNA Polymerase is used for PCR amplification of DNA. Therefore it can not be used for reverse transcription.
It will be a different story if you want to amplify your goi from cDNA. Then, yes, i think you can use Phire polymerase for PCR. As long as you use the same cDNA concentration between your control and sample and amplify them using same cycle, you can get good data for gene expression.
Thank you all! It's indeed a two step RT-PCR so I'm going to give it a try. Thank you again.
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