Any method should work with Sybr green chemistry.  I have used Thermo Scientific's Maxima SYBR green mastermix for microRNA qPCR and it worked well.  For reverse transcription we used the Exiqon miRNA assay primers and RT kit.  In our case we used miRNA standard curves for quantification but there is no reason why you could not use the comparative Ct method if you have a good normalizer in your system.

Dear Prabhakar,

To study the expression of 'mature' miRNAs by qRT-PCR you cannot use the regular primers (and all of the same reagents) that are used for larger-RNAs' detection, instead and if you want to detect by a SYBR-based method, you would need to either add a poly-A tail or ligate a short sequence to the 3' end of miRNAs (to generate a longer transcript). Now you can synthesize the first-strand cDNA using a universal RT primer complementary to the poly-A tail (or to the ligated sequence).

Next, you can quantify miRNA expression by using forward (identical to the miRNA itself) and reverse primers (complementary to the poly-A tail (and perhaps part of the miRNA's 3' end)).

Exiqon and Qiagen assays for SYBR-qRT-PCR-based miRNA detection work good.

And if you want to design primers yourself, that's a different argument (I don't recommend this).

For data analysis, I recommend the comparative Ct method.

Hope this helps.

Sharif

 Yes it is possible

Refrences

http://books.google.com.eg/books?id=Mg44ze0O6lUC&pg=PA177&lpg=PA177&dq=micro+RNA+expression+using+comparative+c+t&source=bl&ots=N0bURyGhX_&sig=d8DZSNB8If65FXrpzIHiSb0Zc5Q&hl=en&sa=X&ei=0shAVLTBNM_kaqTGgugC&ved=0CDAQ6AEwBA#v=onepage&q=micro%20RNA%20expression%20using%20comparative%20c%20t&f=false

http://www.idosi.org/abr/7%284%2913/6.pdf

Guide for relative quantitation real time PCR

http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042380.pdf

Thank you for your valuable answer, 

@Brain, as you mentioned we got 3 normalizer in our profiling study  we will be using those for the expression study using comparative Ct method.

I am interested to know how we can derive miRNA standard, if I am correct, will take reference sample and do the cDNA conversion and do serial dilution, use those for qPCR analysis with respect to normalizer and target miRNA

Thanks

Prabhakar 

I am aware of two possible standards for miR qPCR.  Using the sequence from miRBase, have IDT or other company synthesize the mature miRNA (just make sure you have it purified by RNase-free HPLC to ensure there are no RNases).  As an alternative, duplex miRNA mimics from Dharmacon (they were bought by Thermo then GE) work just as well with the added bonus that they are modified to enhance stability and as such should be stable for years.

We typically setup a 7 point 10-fold dilution standard curve spanning from 100 pg to 0.0001 pg.   You may be able to go above 100 pg but at least for Taqman assays, 0.0001 pg is almost at background.