Ncode miRNA RT & qPCR system - can mRNA be reverse transcribed in same RT reaction? (Feb/16/2009 )
I just got off the phone with invitrogen tech support, and I must say I am quiet disappointed with the response.
I am trying to quantify a miRNA of interest using the Ncode system. I would like to use GAP3DH/B-actin/18s RNA/ 4.5s RNA, etc. as an endogenous control because we dont yet have a good candidate miRNA that could act as a good control.
So I called their tech support and asked if after the poly-A tailing of miRNAs, the Universal RT primer would reverse transcribe mRNAs as well- so I could use the same RT product to run my real-time analysis for the miRNA and a control gene.
The person told me, the Univ RT primer is a oligo-dT with some minor modification. Hence she said it might not work the same as a normal oligo-dT (like in the superscript kits). BUT it does work for 5s RNA since it is small enough to get poly-A tailed. Although I believe her, I would REALLY like to know if someone else has used the Ncode system and quantified 4.5s RNA or other mRNA endogenous controls from the RT reaction products.
Any suggestions are greatly appreciated.
Thanks,
it_never_ends on Feb 16 2009, 12:46 PM said:
I am trying to quantify a miRNA of interest using the Ncode system. I would like to use GAP3DH/B-actin/18s RNA/ 4.5s RNA, etc. as an endogenous control because we dont yet have a good candidate miRNA that could act as a good control.
So I called their tech support and asked if after the poly-A tailing of miRNAs, the Universal RT primer would reverse transcribe mRNAs as well- so I could use the same RT product to run my real-time analysis for the miRNA and a control gene.
The person told me, the Univ RT primer is a oligo-dT with some minor modification. Hence she said it might not work the same as a normal oligo-dT (like in the superscript kits). BUT it does work for 5s RNA since it is small enough to get poly-A tailed. Although I believe her, I would REALLY like to know if someone else has used the Ncode system and quantified 4.5s RNA or other mRNA endogenous controls from the RT reaction products.
Any suggestions are greatly appreciated.
Thanks,
I've been working with the NCODE qPCR system for about a year, and it has been quite a frustrating process. I recently tried my standard b-actin primer set with the cDNA prepared with the NCODE kit, and I did get amplification. So, I suggest trying your standard primer sets (both 5' & 3')--no universal primer. It'll probably work
I was just about gearing up to run the qPCR. I will try out the regular PCR primers as well.
Thanks.
It is probably too late for your experiment, however it is best to use small RNA for the control. You can try hsa- U6, U1, rnu38b, or RNU43 for controls. These may give better results and hold up better to the test of time. If you call Invitrogen they will give you the most recommended control primer sequence. Additionally, if you have room for it you should use more than one primer. Hope that helps anyone looking to similar work in the future.
I am also using this kit now. I separate my total RNA as two parts. One for normal RT to test gene expression, another for miRNA.
I use hsa- U6 and RNU6B as control. They work well.