Identical information provided in the 1st reply by Budak Melayu is available at the "Stratagene’s High-Specificity Primer Design Guidelines" PDF (link below).

https://www.genomics.agilent.com/files/LitItems/miRNA_primer_design_guidelines.pdf

https://www.researchgate.net/post/Can_anyone_help_me_to_design_mature_miRNA_primer_for_qPCR

1)Obtain miRNA Sequence Information

Download the most recent update of mature miRNA sequences (mature.fa "Fasta

format sequences of all mature miRNA sequences") from the Sanger database at

http://microrna.sanger.ac.uk/sequences/ftp.shtml.

Extract the file and then open with Microsoft Word

2)Identify Species Specific Information

Remove all non-specific target species information

For example:

For human sequence (hsa) specific information, delete all non-human miRNA information

3)Align all sequences using ClustalW

Go to http://align.genome.jp

Use default settings

· Push Execute Multiple Alignment (at the bottom of the web page)

· After program has finished alignment

· select clustalw.aln (at top left hand corner of screen)

· aligned sequences will appear

· highlight and copy into a Microsoft Word file

4)Compare Aligned miRNA

· Use the ‘Find’ function in Microsoft Word to locate your desired miRNA.

· You can highlight the desired miRNA sequence in a different color and add blank lines

above and below it to make it easier to see.

· Compare aligned miRNA above and below to the desired miRNA – especially at the

3' end – and identify those with the highest sequence homology.

· Cut and paste those with the highest sequence homology to a separate file.

· You can now close the original file without saving changes.

5)Convert RNA to DNA

· Change the U in the desired miRNA sequence to T (convert RNA to DNA).

· We recommend that you highlight the nucleotides that are not the same as the desired

miRNA in red or some other distinguishable color for easier identification.

6)Identify Nucleotides

· Identify a nucleotide position close to the 3' end that has the lowest nucleotide identity

with the other miRNA – make sure that there is at least 1 nucleotide in the remaining

portion of the sequences that is not the same – delete the nucleotides 3' of this

position.

Note: Nucleotides do not have to be deleted – this is only performed to

increase specificity when needed

· Avoid relying upon a G:T or T:T mismatch for specificity as they are not easily

discriminated

· C:A mismatches are most easily discriminated

7)Calculate Tm

Determine the estimated Tm of the remaining desired miRNA.

Note: You can use the standard Tm calculators of your oligo house. For

example, if using Integrated DNA Technologies website use the default settings to

estimate the Tm.

8)Adjust the Tm

You now need to adjust the Tm of the sequence in order to obtain a Tm in the

appropriate range of 50°C to 55°C. This temperature range provides for greater

specificity.

· Add one or more G to the 5' end of the remaining desired miRNA to adjust the Tm

to 50°C -55oC.

· The minimum length before adding G’s should not be less than 15 nucleotides.

Note: You may determine that there may be more than one sequence for your

desired miRNA primer.

9)Order Sequences

· Design and order your primer(s). Order more if needed. In some cases you will

determine one or more sequences that are projected to provide the desired specificity.

Note: HPLC purification is required for highest consistency and sensitivity

· Order synthetic miRNA templates of the desired miRNA(s) from your standard oligo

supplier and any other closely related miRNA(s). You will want to test empirically for specificity.

Reading the following article in mirbase, helped me put a similar doubt to rest.

http://www.mirbase.org/blog/2011/04/whats-in-a-name/

This is an excerpt of this excellent explanation by Sam, 2011.

The name of a miRNA contains some human-readable information. If you stop reading this post halfway, you’ll likely think this is a good thing. Which of course it is, as long as we recognise the limitations. Hold on to the end and hopefully you’ll see that names can create some issues.

Take for example, hsa-mir-20b. The “hsa” tells us it is a human miRNA. The “20″ tells us that was discovered early — it’s only the 20th family that was named. “20b” tells us that it is related to another miRNA that we can guess is probably called hsa-mir-20a. We can go further — the (lack of) capitalisation of “mir” tells us we’re talking about the miRNA precursor. Or maybe the genomic locus, or maybe the primary transcript, or maybe the extended hairpin that includes the precursor. So that’s already less useful.

hsa-mir-20b has two mature products, named hsa-miR-20b and hsa-miR-20b* (as of this moment — as you’ll see below, this will change). “miR” tells us we’re talking about a mature sequence. In this case miR-20b arises from the 5′ arm of the mir-20b hairpin, and miR-20b* arises from the 3′ arm. The “*” tells us that miR-20b* is considered a “minor” product. That means miR-20b* is found in the cell at lower concentration than miR-20b. It is often inferred that miR-20b* is non-functional, and you’ve probably noticed that miR* sequences in general magically disappear in most pictures of miRNA biogenesis, while the dominant arm is magically incorporated into the RISC complex.

If you are checking the expression of the mature miRNA, and you know the sequence of the mature miRNA then use the seqence(s) of the mature miRNA, regardless of 5p or 3p, if both express in you tissue then you shold (try) to detect all of them. Also a possibility , if you dont care about the 3/5p and there is a possibility, try to choose the primers that anneal on both (in some cases the mature miRNA sequence is very similar). I would suggest, to first figure out which specific mature form is relevant for you (literature, expression studies) and then focus on them. Or you can first pick the most interesting ones and then the rest, because i would suspect it will be quite difficult to properly conduct expression study on the several miRNA with 'home-made' primers.

At the end it might be cheaper to order pre-prepared probes for expression assays, because you don't need much optimization (in most of the cases)

Thank you Budak kuantan for this nice explanation. I will try with these steps and information.

Identical information provided in the 1st reply by Budak Melayu is available at the "Stratagene’s High-Specificity Primer Design Guidelines" PDF (link below).

https://www.genomics.agilent.com/files/LitItems/miRNA_primer_design_guidelines.pdf

Can anyone used the following programme before:?

http://sourceforge.net/projects/mirprimer/?source=typ_redirect

how can i synthesize the universal cDNA of miRNA 

Saied Afshar, you can use good quality mirna based product such as EXIQON miRCURY LNA™ Universal RT microRNA PCR and convert your all mirna from total rna to cDNA.

@ Deilson Elgui de Oliveirae I am trying to get an access to the link that you have mentioned above, but it is not opening. It is therefore requested to please send the protocol for miRNA primer designing. I will be grateful for your this act of kindness.

Thank You.