I think this document can help you. I use the mirScript system (Quiagen) and use the miRNA sequence as a primer. Primers synthesized by any company can be used.

https://pure.uva.nl/ws/files/2312070/127153_08.pdf

I presume you're referring to the stem-loop RT system and not a ligation-dependent method. If so, you need RT primer sequence followed by sequences for forward and reverse primers and a probe. A place to start if you want to try your own would be the original paper by Caifu Chen and colleagues (http://www.ncbi.nlm.nih.gov/pubmed/16314309 ), which included a supplement with stem-loop RT primer sequences (for the RT step) plus fwd and rev primers and hydrolysis probes for several miRNAs. It's not clear whether the company is using these exact sequences in its current assays, and one might suspect that at least some of the non-miRNA-complementary portions of the stem loop primers might have been optimized for specific miRNAs. But as I said, a place to start. Several groups have developed similar methods; search "real-time quantification" "microRNAs" "stem-loop RT-PCR" on PubMed.

I am not sure if it is stem-loop RT or modified oligo(dT) primer method.

I am using:

1. MiRNeasy Mini Kit for RNA isolation from my cells

http://www.qiagen.com/products/applications/mirna-research/purification/mirneasy-mini-kit?applicationflowStep={C7348BEB-824C-4597-BCB8-91A2DD4EC0C4}&StepName=Purification&Page=2

2. miScript II RT Kit to get cDNA from my RNA

http://www.qiagen.com/products/applications/mirna-research/analysis/miscript-ii-rt-kit?applicationflowStep={C7348BEB-824C-4597-BCB8-91A2DD4EC0C4}&StepName=Analysis&Page=2

And then the company wants you to use the:

3. miScript SYBR Green PCR Kit which has the qPCR materials plus a universal reverse primer for the microRNA detection

http://www.qiagen.com/products/applications/mirna-research/analysis/miscript-sybr-green-pcr-kit?applicationflowStep={C7348BEB-824C-4597-BCB8-91A2DD4EC0C4}&StepName=Analysis&Page=2

4.miScript Primer Assay which is just the forward primer to use with the master mix

http://www.qiagen.com/products/applications/mirna-research/analysis/miscript-primer-assays?applicationflowStep={C7348BEB-824C-4597-BCB8-91A2DD4EC0C4}&StepName=Analysis&Page=2

So I already have 1 and 2, but instead of using 3 and 4 from qiagen and spending a lot of money we want to use our own SYBR Master mix, add in our own universal primer (which my supervisor found the sequence to) and then add in our own designed forward primers which we would synthesis for $10 instead of over $80.

I have the sequences of the miRNA I want to test, so can I use some sort of primer design software to make the forward primer for each of my candidates?

Ya I am worried about that too as I don't have an unlimited amount of time in my masters. But I found this from another question on researchgate:

For making cDNA, miScript II and other products add a poly A tail to the microRNA. During cDNA sythesis the complementary cDNA version of the miRNA is made with an oligodT adapter attached at the end. The reverse primer in the RT-PCR is then a universal primer for the dT end, which is generally provided in the SYBR kit, while the forward primer is the DNA version of 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.

One additional factor that needs to be considered 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.

Ah, this method is neither stem-loop nor ligation. A question, though: when the forward primer is simply the "DNA version of the miRNA" and there's no probe, what provides specificity for mature miRNA? Adding a few A's might help, but how many are enough? To balance Tm, additional A's could mean compromising discrimination between miRNAs with similar 3' sequences. The ligation methods can have similar issues, but at least there you could have something more complex than poly-A on the end of a mature sequence.

I've actually used the method Nathan Johnston proposed, with success. But do check for the existence of closely related but different miRNAs or you're going to run into problems with specificity. We always used the same standard PCR program for all miscript style qPCRs, Qiagen or self-designed.

When using Qiagen miScript and the method described by above by Nathan for primer design, what is the optimal Tm to aim for?

Well the SYBR qPCR protocol from Qiagen uses an annealing temp of 55C i believe. So probably around 59C. It all depends on what your qPCR protocol states for the annealing cycle and then you can add 3-5C to determine your target Tm.

what about the 3 ' dgenerate anchot of the reverse transcription primer of qiagen this means that not poly ttttt tail only are attached to tha cdna of the mirna but there is a separating degenerate anchor????

I was reading this very interesting paper from Dr Busk: 

http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-11-70

To me it looks like the same protocol used by QIAGEN, the main issue is that the tag sequence used with the RT-primer is proprietary, thus it is not possible to design the reverse primer custom. Maybe one could try with the universal primer sold with the SYBR mix by QIAGEN and just design the forward primer manually (would still save $50 per primer pair). Did anyone try it? Does it work?

Nathan, have you tried to design the primers with the method above? Did you have success in your qPCR experiments?

I think this document can help you. I use the mirScript system (Quiagen) and use the miRNA sequence as a primer. Primers synthesized by any company can be used.

https://pure.uva.nl/ws/files/2312070/127153_08.pdf

For canonical miRNA sequences, miRBASE:

http://www.mirbase.org/

Some old protocols use the miR sequence as forward primer but this does not confer sufficient specificity to the assay. You are not even able to assess what you have amplified as the primer has the same sequence as the miR.

miR-specific qPCR with two, specific primers and a universal RT primer gives high specificity ( Article Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

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

Article Quantification of miRNAs by a Simple and Specific qPCR Method