4 replies to this topic

[preston]

Hello, I'm French and I apologize in advance for my English.

We currently have a problem with our qPCR on miRNA. We use the kit nCode miRNA First-Strand cDNA Synthesis for polyadenylation and reverse transcription of miRNA. For qPCR we use our own SYBR Green and our own protocol.
We tested several quantities of miRNA, ranging from 0.5 to 2 µg. The primers used are universal primer (complementary to the poly-A) and a primer complementary to our miRNA. Only during the PCR, our samples do not come out, there are only the baseline. Even adding 10 cycles with our PCR cycles to reach 55 total. We also tested several Tm and several primers miRNA but nothing has changed.
We ask ourselves the question: how to determine the quality of our cDNA?
After extraction of our total RNA, we found their quality with Agilent Bioanalyzer 2100, samples used with a RIN greater than 7.
If anyone has encountered this kind of problem, its help will be welcome.
Thanks.

Edited by preston, 15 March 2011 - 03:15 AM.

[Fizban]

preston, on 15 March 2011 - 03:14 AM, said:

Hello, I'm French and I apologize in advance for my English.

We currently have a problem with our qPCR on miRNA. We use the kit nCode miRNA First-Strand cDNA Synthesis for polyadenylation and reverse transcription of miRNA. For qPCR we use our own SYBR Green and our own protocol.
We tested several quantities of miRNA, ranging from 0.5 to 2 µg. The primers used are universal primer (complementary to the poly-A) and a primer complementary to our miRNA. Only during the PCR, our samples do not come out, there are only the baseline. Even adding 10 cycles with our PCR cycles to reach 55 total. We also tested several Tm and several primers miRNA but nothing has changed.
We ask ourselves the question: how to determine the quality of our cDNA?
After extraction of our total RNA, we found their quality with Agilent Bioanalyzer 2100, samples used with a RIN greater than 7.
If anyone has encountered this kind of problem, its help will be welcome.
Thanks.

May I be blunt? try to use the kit protocol for PCR! You introduced an unwanted change in something that should have already been set up by the producer of the kit. why did u change it? Do u have control RNA? Have u tested your RNA with other miRNA qPCR systems (like applied biosystems for example?) did u try RT-qPCR for gene expression? If RNA is good your system is not working. you could either decide to make it work or to change system.

[UBClabbie]

preston, on 15 March 2011 - 03:14 AM, said:

Hello, I'm French and I apologize in advance for my English.

We currently have a problem with our qPCR on miRNA. We use the kit nCode miRNA First-Strand cDNA Synthesis for polyadenylation and reverse transcription of miRNA. For qPCR we use our own SYBR Green and our own protocol.
We tested several quantities of miRNA, ranging from 0.5 to 2 µg. The primers used are universal primer (complementary to the poly-A) and a primer complementary to our miRNA. Only during the PCR, our samples do not come out, there are only the baseline. Even adding 10 cycles with our PCR cycles to reach 55 total. We also tested several Tm and several primers miRNA but nothing has changed.
We ask ourselves the question: how to determine the quality of our cDNA?
After extraction of our total RNA, we found their quality with Agilent Bioanalyzer 2100, samples used with a RIN greater than 7.
If anyone has encountered this kind of problem, its help will be welcome.
Thanks.

How did you isolate your RNA? Are you using total RNA or small RNA?
miRNA are very short, only ~22 nt long. So your RNA isolation method could adversely affect whether you retain miRNA or not. I always have reliable results using Trizol to isolate total RNA. There really isn't any need to isolate small RNA if your mastermix formulation and qPCR program are reliable.

It seems as if something might be wrong with either your RNA sample (like I stated above) or with your SYBR Green mastermix. Your mastermix is probably formulated for much longer sequences. Have you run a control on some non-miRNA sequence? Try an endogenous control like U1. It doesn't make sense that you aren't achieving any signal at all. The more likely problem is the exact opposite, is achieving signal when you should't due to contamination or non-specific binding.

many miRNA species differ by only 1 nt. So even if you are targeting low copy number miRNA you should still get a later signal around 40 cycles from sequences differing by 1-3 nts.

[preston]

Thank you for your answer!
It turns out that our SYBR Green is not specific enough of our miRNAs.
We seek to use another SYBR Green, or another fluorescent agent.
We would like to avoid using Taqman probes.
Someone knows a specific miRNA product and has already used?
Thank you in advance!

[Earnest]

preston, on 24 March 2011 - 03:03 AM, said:

Thank you for your answer!
It turns out that our SYBR Green is not specific enough of our miRNAs.
We seek to use another SYBR Green, or another fluorescent agent.
We would like to avoid using Taqman probes.
Someone knows a specific miRNA product and has already used?
Thank you in advance!

I've used these before:

"http://abmgood.com/miRNA(microRNA)/miRNA-qPCR-Mastermixes.html"  They work great on my Roche Lightcycler 480 II and much older ABI...  

They have specific formulations for different machines, so you'll have to match your up.  They recently sent an email promo flyer out for 5 mL free mastermix to promote formulations for the newest machines - call them up and ask, or sign up for an email subscription - I think the promo may still be on, though you do have to pay for shipping.

Edited by Earnest, 24 March 2011 - 09:15 AM.