I've been wondering if removing the random primers from the reverse transcription reaction either with a column or precipitation before using it as a qPCR template reduces the background a bit in later cycles.
Notes:
1. I am working with bacteria so some amount of genomic DNA amplification is inevitable no matter how much I DNase treat it.
2. I am using the BioRad iScript kit for reverse transcription.
3. I am quantifying cycle thresholds with a real time PCR machine using Sybr Green.
Hello Alexandra Johnson ,
Theoretically, if you remove the non-target cDNA, it could possibly improve the sensitivity of the reaction, but I think that improvement will be negligible.
In practice, if you eliminate non-target cDNA (after RT), either by column, magnetic beads, or by precipitation, you will lose concentration of the total cDNA. This can worsen the sensitivity of PCR.
An alternative option is to do the RT with specific primers instead of random primers. That would make your total cDNA correspond only to the target of your PCR, and no purification of the cDNA would be necessary. Also mention that it is very important to treat the total RNA extracted from the bacteria with DNAse.
if my answer has been useful, please recommend it.
:) :)
Good luck and Merry Christmas Alex!
I would suggest you to use magnetic bead clean up. In column or precipitation based cleaning you will loose a lot of your product.
Hello Alexandra Johnson ,
Theoretically, if you remove the non-target cDNA, it could possibly improve the sensitivity of the reaction, but I think that improvement will be negligible.
In practice, if you eliminate non-target cDNA (after RT), either by column, magnetic beads, or by precipitation, you will lose concentration of the total cDNA. This can worsen the sensitivity of PCR.
An alternative option is to do the RT with specific primers instead of random primers. That would make your total cDNA correspond only to the target of your PCR, and no purification of the cDNA would be necessary. Also mention that it is very important to treat the total RNA extracted from the bacteria with DNAse.
if my answer has been useful, please recommend it.
:) :)
Good luck and Merry Christmas Alex!
If we remove the complementary DNA which is that target sequence .it would be affect the cycle..so use the PCR
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