Ncode qRT-PCR - (Sep/21/2009 )

Hi

I'm just starting to work with microRNAs, and starting also to have problems! I just want to know the level of expression of some miRs in some lymphoid cell lines.

For profiling, I ordered NCode qRT-PCR detection kit, from invitrogen because in this kit they use a universal primer to do the cDNA, so I wouldn't have to make a different cDNA with miR specific primers for each one I want to detect. I designed the primers specific for the miRs qRT using invitrogen tool http://escience.invitrogen.com/ncode. . As they say in the kit that enrichment for small RNAs it's not necessary, I 1st extracted total RNA with TRIZOL.
For all miRs I got inespecific bands when i run the qRT-PCR in the gel. Besides the band corresponding to the miR, I got one around 150-180, depending.
So I ask invitrogen for support, and they recommend to design new primers, beside the fact that I ordered then from their database. they strongly suggest to modify the primers to get a melting temperature of 60oC. I wondered why they didn't put them already with the proper characteristics in their database!!!!

I don´t know if this is going to solve the problem because the primers already encompass all mature miR sequence, so, I would just put random bp to increase TM, but I don't know if this would increase specificity..... what do you think?

tks in advance

Sounds like they're system isn't that great if you need to modify their "pre-approved" primer sequences with random nts. Not sure how that could help specificity. Additionally, if you use a universal primer, how do you amplify the mature miR? I think you'll only be amplifying the pri/pre-miRNA?

Depending on which mature miRNA you want to detect, sometimes you have to "enrich" or isolate small RNA by using miRNA purification kits.

If the miRNA detection kits use polyA polymerase to add AAAA to the 3' end of mature miRNA, you might get different length of qPCR products because the polyadenylation time varies.

If the enrich small RNA still not working with SYBR Green qPCR, then you might want to switch to Taqman.

Hi Nadia,

I am also facing the same problem. I get an additional band at around 130bps. We think that the kit probably also amplifies precursor form of the miRNA and hence the 2 bands on the gel.

Shall keep u posted on my developments.. :-)

miRNA_diabetes on Oct 23 2009, 01:34 PM said:

I think your 130bp band should be the right one. Your kit should not amplify the pre-miRNA.

Hi Nádia.c and miRNA_diabetes,
I was wondering if you any new information about your problem. When I read your posts I just couldn't believe it because not only we are sharing the same problem but we are sharing the same views about their database http://escience.invitrogen.com/ncode.

We extracted total RNA with TRIZOL and purified it with Norgen column. We tried 3 miRNA we ordered from the database with Ncode kits and ran the qPCR products on a gel and got either multiple bands or 1 band around 130-150, only one seemed to have worked so I rang invitrogen and they told me I needed to design new primers to get a melting temp of 60C (just like you!!!!). So we did and I just tried new primers at 60C and still doesn't work i.e same pattern as before 1 band around 130-150 and the other is like yours i.e 2 bands (one band corresponding to the miR so a bit of improvement, the other one around 130-150).

Not sure what to do next.

I don't know if this discussion is still active if yes please let me know if you have progressed.
Cheers
Diane

This is going to sound bad but I'm happy that someone else is having the same problem, because I really think - and you confirm to me - that the problem is the Ncode system and not us or our hands. I simply gave up of this system and I'm not going to use it anymore. I 'm using now the primers from exiqon, because they are LNA primers, much more specific.
Glad you send the email to me, because I never saw the topic again.
good luck with your experiments and spread the word

cheers

Nádia