I don't think cDNA purification should always be performed. Actually, I've done quite a lot of cDNA synthesis followed by quantitative PCR, and have never purified cDNA. However, I do dilute my cDNA prior to qPCR between 2 and 10 fold dilution depending on the expected amount of template you want to amplify. In fact, if your concern is the low specificity you could get from amplification, then the main issue is to design good specific primers to your template.
Yes, I strongly recommend for cDNA purification prior to RT-PCR, because the enzymes in cDNA synthesis will hinder the PCR, especially polymerase activity.
Depends on what you need require after RT-PCR, as in reverse-transcription and not quantitative PCR. If it's just a routine PCR to get some genes (e.g. get some reference gene sequences for cloning/sequencing), then it does not matter.
However, it it is to generate novel sequences, quantitative PCR etc, then yes you should do clean-up.
Edit: Sorry I just read more of your post. You're talking about quantiative PCR, then yes you should do clean-up. In fact, run tests with reference genes and make standard curves etc to check the quality of your template. Also run the RIN quality check (ThermoScientific) if possible.
Thank you so much for your help! Ill try my best to incorporate these suggestions! Cheers.
I don't think cDNA purification should always be performed. Actually, I've done quite a lot of cDNA synthesis followed by quantitative PCR, and have never purified cDNA. However, I do dilute my cDNA prior to qPCR between 2 and 10 fold dilution depending on the expected amount of template you want to amplify. In fact, if your concern is the low specificity you could get from amplification, then the main issue is to design good specific primers to your template.
cDNA purification has nothing to do with SYBER specificity, as advised above the reason might be less specific primers
You don't have to purify cDNA for qRT-PCR. Just design very specific primers. You can dilute your cDNA enough for your plate or number of genes you want to test. In case of non-amplification or no detection, you can use more cDNA. Remember when you clean your cDNA you loose a lot. Best!
I have conducted RT-PCR experiments after cDNA synthesis, but never purified it. I diluted the cDNA (1:100) before using in the reaction with well-designed primers targeting to my gene of interest. In some cases as with enzymes, dilution is not necessary as well. So its not a issue. Consider the purity and quantity of Total RNA before cDNA synthesis.
Thank you for your answers every one! I am relatively new to to RT PCR processing that's why i get easily mislead by products in the catalogue . Thank you because Now I don't have to spend unnecessary bucks on unnecessary reagents..
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