will rna get amplified when using it as a template for PCR?
-using dna specific primer
-with gc% 55
-using high fedility polymerase.
Hi,
No, uracil stalls polymerases, especially a high fidelity one . You should reverse transcribe your RNA into DNA first
Hello
You can use a total RNA as a template of PCR. You should replace the random primer (or oligo DT) of your cDNA kit for your specific primers (see your cDNA kit specifications).
High fidelity cDNA synthesis Kit or One-step RT-PCR they have a high fidelity polymerase. Make sure if your enzyme belongs to this Kits.
If you don't have this high fidelity kits, the best option is transcribe your RNA into cDNA first, than do the PCR.
Dear Nithin,
Your reaction will not amplify RNA since:
- RNA requires uracil residues
- You would need RNA polymerases in your reaction mixture
Abhishek
Thanks, but the problem iam facing is even though i treat my rna sample with dnase i get amplification with the treated rna. Even the spec show 260/280 as 2.
Please clarify your experimental design, since you are treating your template with DNAse and using DNA specific primers. Theoretically RNA cannot be amplified without RNA polymerases, can you delineate the high fidelity polymerases you are using?
Hello Abhishek,
My aim is expression analysis. I have isolated the total RNA from bacteria(RNAeasy mini kit) and then treated with Dnase(ambion). Now to cross check their is no DNA contamination before cDNA conversion. I used the Dnase treated RNA as template and amplified my DNA specific primers and i got positive bands. I did not know why.
One thing i can do is by increasing the amount of Dnase. Let me try that. If you have any other suggestion kindly share. Thank you.
Hi Nithin,
I am now facing a similar porblem. I have designed the qPCR primers in the exon-exon junction, and even if I DNase treated my samples, my NRT is positive. How you found what your problem was?
Hello Floriana
I believe if the CT value is above 35 we can neglect it. For instance my Positive RT was around 25 while my negative RT is around 35. It is very difficult to remove the entire DNA.
Since you have mentioned your primer is designed in exon-exon junction you can try reducing the extention time, may be that can help. In my case it was prokaryote so could not do it.
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