In general, what Ashwani suggested work well.

One additional factor that needs to be consideedr is to match Tm between forward and reverse primer. Using the following guideline that my lab has been using, we successfully designed and validated ~200 microRNA qPCR using SYBR.

To increase Tm

1st Add “A” at the 3’ end of primer sequence.

(We are adding “A”s, since we did poly “A” tailing and now a stretch of “T”s are at the end of cDNA that was made from the mature miRNA.)

No more than 6 "A"s should be added to any one sequence.

2nd IF an increase in Tm is STILL needed, G or C residues can be added to the 5’ end of primer.

Try be less than 30 nucleotides

To decrease Tm

1st Delete sequences from the 5’ end.

However, the resulting primer should be still at least 18-mer match to the target miRNA.

2nd In MOST cases, bases can be also deleted from the 3’ end.

But care SHOULD be taken to analyze the best position to truncate WHEN there are closely related family members.

PS) We now published our miR qPCR primer sequences at the following paper. If you use, please cite this paper. Please note that the performance of qPCR varies depending on samples. For example, with certain qPCR assay, we can get reliable data using brain tissues whereas failed to get good data using CSF.

http://www.nature.com/articles/s41598-017-13031-w

Article Intra- and Inter-individual Variability of microRNA Levels i...

if you have mirna sequence then ........

you can go with primer3, one of best tool for designing primer for pcr. go through this link http://simgene.com/Primer3

set your parameter according to your requirement and choose one of them try it.

best of luck.... :)

Thanks Chintan...But the problem is mature mirna sequence is about 20 bp. so any software is not available to design this kind of primer

For qPCR of mature miRNA, a different technique known as stem-loop RT-PCR has been shown to be able to specifically detecting the mature miRNA. Try look into articles regarding this technique in PubMed.

Dear all,

What will be advisable, designing primer to mature miRNA sequence (20-24bp) or to miRNA precursor sequence ( more than 50bp)?

For making cDNA ,miscript II and other product from system biosciences (quantimiR) adds poly A tail to the microRNA. By attaching oligodT adapter it introduces universal tag .The reverse primer in the rtpcr is universal primer generally provided in the kit while the forward primer is the mirna which you are interested in. So it is very easy to design the primer just replace the U's with T in mature miRNA sequence i.e. make it a dna sequence and you have a forward primer . For detail you can read the manual of the miscript kit it is available in the qiagen website.

In general, what Ashwani suggested work well.

One additional factor that needs to be consideedr is to match Tm between forward and reverse primer. Using the following guideline that my lab has been using, we successfully designed and validated ~200 microRNA qPCR using SYBR.

To increase Tm

1st Add “A” at the 3’ end of primer sequence.

(We are adding “A”s, since we did poly “A” tailing and now a stretch of “T”s are at the end of cDNA that was made from the mature miRNA.)

No more than 6 "A"s should be added to any one sequence.

2nd IF an increase in Tm is STILL needed, G or C residues can be added to the 5’ end of primer.

Try be less than 30 nucleotides

To decrease Tm

1st Delete sequences from the 5’ end.

However, the resulting primer should be still at least 18-mer match to the target miRNA.

2nd In MOST cases, bases can be also deleted from the 3’ end.

But care SHOULD be taken to analyze the best position to truncate WHEN there are closely related family members.

PS) We now published our miR qPCR primer sequences at the following paper. If you use, please cite this paper. Please note that the performance of qPCR varies depending on samples. For example, with certain qPCR assay, we can get reliable data using brain tissues whereas failed to get good data using CSF.

http://www.nature.com/articles/s41598-017-13031-w

Article Intra- and Inter-individual Variability of microRNA Levels i...

Some companies sell the universial reverse primer at a ridiculously expensive price. While regular primers should be around $3 - $4 , these companies charge ~$100. Using a simple molecular biology technique, you can easily sequence this universial reverse primer and order it at $3 - $4.

Dear Jungsu Kim,

Please elaborate your answer, how to select this universal reverse primer?

I have the same problem: I'd like to design and order primers for miRNA qPCR from local company instead of buying it from Qiagen. So do you think that is it a good way to clone PCR product, sequence it and get primer sequences?

Yes, you can clone the PCR product and then sequence it.

TA vector will work well for this purpose.

I don't know whether this violates any law, though.

For qPCR, you can follow the design made by Caifu Chen in 2005 (http://nar.oxfordjournals.org/content/33/20/e179.abstract). It is based on the idea of having a stem-loop primer to amplify only mature microRNAs. Another option is to buy them directly from Invitrogen.

MicroRNAs are 22-24 bases long so its possible to design 12-18 nucleotide long forward primer and reverse primer, 3-8 specific nucleotides at 3' end and extend it with universal tag, for c-DNA synthesis.    

Hi Dipta,

I used with success a software to design primers for miRNAs. Here is the paper describing the software:

http://www.biomedcentral.com/1471-2105/15/29

Good luck with your work.

Pierre

I found one more useful web tool here:

http://www.leonxie.com/miRNAprimerDesigner.php

Does anyone used Invitrogen Kit- NCodeVilo for miRNA cDNA synthesis? Any reviews?

I used that kit ankita..its nice and its give publishable data

Were you guys able to design a reliable primer for the endogenous control U6 small RNA for human samples?

miRNA Primer Design:- 

1. Open miRBase, enter interested sequence.

2. Open respective miRNA & fetch the Sequence.

3. Alongside, open the miRNA Primer Design Tool & login in that software.

4. Enter the miRNA sequence in the box & select URL probe.

5. Enter the design option.

6. select the Universal reverse & forward primer according to your convenience. 

Hi,

Based on my experiences, it will be very helpful if you can design and use stem-loop primer for the cDNA synthesis. There are lots of interesting works which suggest and provide successful designature of stem-loop structure that can be customized according to your desired miRNA. Use the keywords such as stem-loop primer, qPCR and miRNA. you will surely find the optimized methodologies.

Hi, I worked as a research scientist in a project where I was engaged in primer design for miRNA by using quantitative PCR-TaqMann. You can take a look to the following papers as I did:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1292995/

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115293

https://www.ncbi.nlm.nih.gov/pubmed/21732315

I hope they help.

Hello Dr. Dipta Sengupta

i suggest you these two papers:

1- MicroRNAs Specific Primer Design using miRNA Design Tool

http://www.isca.in/IJBS/Archive/v6/i8/6.ISCA-IRJBS-2017-061.pdf

2- A tool for design of primers for microRNA-specific quantitative RT-qPCR

Article A tool for design of primers for microRNA-specific quantitat...

good luck

MicroRNAs Specific Primer Design using miRNA Design Tool : http://www.srnaprimerdb.com/

Using R

https://github.com/caanene1/miRNAprimer