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

Hi,

in this link, you can design your primers

http://www.mirbase.org/search.shtml

As a regular RNA based in Mirbase data of pri-miRNA

Hello,

I have a good experience with the miSCRIPT Qiagen kits for RT-qPCR of miR. I use it with my own design for each miR targeted : I choose the forward miR - specific primer as all or a portion of the miR sequence recovered in miRBase, and it works with an universal reverse primer provided in the kit (unknown sequence obviously) ; the product is always around 100 pb according to electrophoresis migration.

The kit provides a RT buffer which allows reverse transcription of all kinds of ncRNA ( HiFlex buffer).

I think that if you design correctly your gene-specific forward primer from the sequence found in miRBase, you shoud be able to quantify specifically your pri-miR : check carefully the specificity by BLAST.

Best regards,

Claudine

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

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.