2 replies to this topic

[Panini]

HEllo everybody,
I have a lot of experience in qRTPCR but I need some back up voice. Recently I moved to a new lab where Oligo dT is used in the RT step. But 18s is used as endogenous control. Its SYBR green PCR and there is no DNAse treatment. My take is that a. you cannot use 18s as endogenous control because you are using Oligo dT and b. the amplification you see is probably DNA.
Am I right or wrong, do I have much much more to learn about qRTpCR or is there a "mRNA" population for the 18s rRNA that can be specifically amplified using the "right primers"??
Please give your opinion.

[zhongmindai]

Panini, on 06 May 2011 - 03:43 PM, said:

HEllo everybody,
I have a lot of experience in qRTPCR but I need some back up voice. Recently I moved to a new lab where Oligo dT is used in the RT step. But 18s is used as endogenous control. Its SYBR green PCR and there is no DNAse treatment. My take is that a. you cannot use 18s as endogenous control because you are using Oligo dT and b. the amplification you see is probably DNA.
Am I right or wrong, do I have much much more to learn about qRTpCR or is there a "mRNA" population for the 18s rRNA that can be specifically amplified using the "right primers"??
Please give your opinion.

Some rRNAs may be polyadenylated for their decay, so it is possible to use Oligo-dT in RT to reverse transcribe these rRNAs. However, I am not sure if these rRNAs could be used as endogenous control as themselves may be differentially regulated.

Edited by zhongmindai, 08 May 2011 - 05:48 AM.

[Trof]

18S doesn't have polyA tail.
You can check the gDNA contamination by doing a -RT reaction.

This paper suggest using oligo dT and 18S specific RT primers to coamplify 18S with mRNA.