I designed the primer sets for miR-625 , but i have the problem with C-DNA synthesis for real-time PCR. i need the protocol of C-DNA synthesis .
Is there anyone to help me about the reagents ,time of reaction and thermal cycler program?
Hi Afshar,
For the conversion of miRNA specific cDNA, you can use a special kit provided by Applied biosystems. The method involves a stem loop primer which converts the mature miRNA from the total RNA pool into miRNA specific DNA which can later on be analysed by TaqMan based miRNA assay system.
The thermal profile of cDNA conversion is as follows:
16C for 30 min followed by 37C for 30 min followed by 85C for 30 min and a final halt at 4C
The cDNA so obtained is further diluted before setting the Real time qPCR reaction.
Hope you find answer to your query.
with best regards
Saeid,
cDNA conversion kits normally poly-adenylate the total RNA pool which allows for all RNAs to be converted to cDNA. You can then use targeted miRNA premiers for a qRT-PCR protocols.
As mentioned above applied biosystems sell a kit and protocol just for this purpose.
Dear Afshar, I can also only recommend to use a kit, it is quite troublesome to do it without. We successfully used components from Genecopoeia (http://www.genecopoeia.com/product/mirna/) - they have sometimes nice offers.
regards- Kai
Hi Saeid,
I can recommend this reverse transcriptase kit that my lab uses to make cDNA of miRNAs and then proceeds with PCR as usual and we get good results (it also works for mRNA). From Qiagen: https://www.qiagen.com/us/search.aspx?q=218161#&&pg=1
Hi Saeid,
Theoretically you can use any kind of reverse transcriptase you prefer, just changing the cycle for the synthesis. I follow the protocol described at this paper:
Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP: A highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 2007, 3:12
Good luck.
You can use any cDNA synthesis kit because it allows synthesis from total RNA and use specific primers for miRNA in PCR. Even you can use miRNA specific primers to synthesize cDNA. Kit is good option
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