Hi Giuseppe..

Please see the attached reference...

You can also consider stem-loop primer method. It's more economical and still specific. See this:

http://nar.oxfordjournals.org/content/33/20/e179.short

We've been using the Applied Biosystems stem loop assays for years with excellent success.

I like taqman primers. They are very specific and require very less quantity of total RNA.

Taqman is the best if you have a short list of miRNAs you want to go after, or have a larger list you could make a qRT-PCR array plate from. Otherwise, Qiagen works okay, but I question the specificity moreso than Taqman. At least with Qiagen, you don't need to know which ones you're going after before you make up your cDNA.

Check this out:

http://www.exiqon.com/mirna-pcr-primer

I would like to mention a serious flaw in believing in miR TaqMan assay without using proper negative control. While TaqMan may appear to be more specific given its unique design, things do not work as you expected in real experiments. We had cases where miR TaqMan assays give no signal with no-template control (H2O) and give very strong signals with synthetic miR in H2O, BUT they also generated very strong signals with RNAs extracted from microRNA knockout mice tissue. With complex real biological samples, certain miR TaqMan assay clearly gives false non-specific signals from miR KO mice. In these cases, I would not trust any signal from TaqMan with real biological samples.

Taqman (Applied Biosystems) worked for me

Try ncode™ vilo™ mirna cdna synthesis kit and express sybr® greener™ mirna qrtpcr kits from invitrogen

I consider LNA to be a more expensive technology. What I can suggest you is from Origene.

The kit will first polyadenylate the 3' end and the use an Oligo DT primer to amplify.

Please visit the below page:

http://www.origene.com/geneexpression/qpcr_mirna/

this one as well from ambion: (miRvava)

http://products.invitrogen.com/ivgn/product/AM1558?ICID=search-product

Good luck!

Gaurab

Fro the new comers, it is best suggested that, be patient, cool, read the instructions carefully and reapeat your experiments

Samir Kumar Patra, Professor of Biochemistry, Mol Biol and Epighenetics

I'm using Applied biosystem for 5 years and it works fine

Xavier nissan

I have used exiqon's kit for mature miRNA amplification (both cDNA synthesis,SYBR green and primers ) and worked fine. But they are little expensive compared to qiagen.

For most cases I would, like the majority, recommend the TaqMan microRNA Assays from Applied Biosystems. It is a bit more expensive than the others but it is also, in general, more specific. The only downside with using that assay is, as people mentioned before, that you have to do the RT-PCR using the microRNA specific primers, so if you after running your first samples realize that you would like to check another microRNA as well, you have to start from scratch again.

I use the snoRNA202 as my endogenous control for short RNAs, and that always works fine. Also I suggest, like previous speakers, that you should have a negative control = water instead of RNA/cDNA, and if possible also a positive control from samples/tissues where you already know that the specific microRNA is expressed. If it is the first time that you try the assay, it could make sense to try making a standard curve as well to see that the assay works like it should.

Gaurab Banerjee mentioned LNA, locked nucleic acids. We use these oligos as probes when doing northern blots for short RNAs with good results, so if you later would like to verify the qPCR results with a northern blot, I would strongly recommend these probes from Exicon

Check the stem-loop RT-PCR method by ABI;

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1292995/?tool=pubmed

QPCR worked well for us...I think. Stil concerned about pre-pri vs mature levels not being caught, but Liang Chu in our lab McDonnell 510 is expert at least for larger ncRNAs

My PI has done a bit of investigation and says that reports on the miRvana kit are excellent---it seems to be the miRNA isolation kit used in a large number of published miRNA studies. Our lab has had some success with the miScript SYBR Green II PCR kit from Qiagen: http://www.qiagen.com/products/miscriptsybrgreenpcrkit.aspx, using the Primer Assays provided by Qiagen, as well.

The TaqMan kits for miRNAs from AppliedBiosystems have worked well in our hands. We have used these kits extensively, and found good correlation between RT-PCR results and microarray results.

Dear researchers,

Necessary to refine the research to be done? Cell Line, plant, tissue or animal testing?

I have a protocol that might help you.

Regards,

Here I add the file to assess the expression of miRNA

I used the System Biosciences (SBI) kit, and I would say is cheap but is not a good kit, since I got too much variability on technical replicates due to the oligos. I tried the Exiqon kit and I think it is very good, a bit expensive, but very good!

TaqMan MicroRNA Assays Protocol would be an excellent choice

https://genome.med.harvard.edu/documents/qpcr/microRNATaqManAssayProtocol.pdf