can you please help me to design primers for reverse transcription of miRNA and - miRNA primers (Aug/25/2010 )
I am VERY new to the field of miRNA. I am interested in synthesizing cDNA from miRNA derived from total RNA (trizol extracted DNAse I treated). I normally use AMV kit from Roche to sythesize cDNA from RNA. Now for cDNA from miRNA can I still use same kit or do I need a special one. Also what primers would I need if I wanted to amplify miRNA can I use random hexamers or are they no good? Do I have to use miRNA specific primers? If so can you give me exact details of how to design or if there is an online program?
Second question I have is the method for quantification. Normally for cDNA I performed sybr green using two gene specific primers that are 20 bp and generated from primer 3 program (online). What primers do I need for let's say miRNA: 3' cuaAGGUUAAAAAGGUGUAGAa 5' hsa-miR-576-3p??? When they say loop stem primers what exatly do you mean? How do I find the where is the loop portion? Is there a program to help me out?
Any ideas/ advice will be greatly received!!!!
If you want to determine the expression of the mature miRNA then I think you'll have to use TaqMan assays. Otherwise the miRNA (~22nt) is too short to amplify. The TaqMan assays use the stem loop primer - check Fig 1 at ABI here: https://products.appliedbiosystems.com/ab/en/US/adirect/ab;jsessionid=rQhmM2XQ6q1HLC8mhKmj3SzcJnTS6SPh1qngbrs15QwGpK1ZTfRX!645065806?cmd=catNavigate2&catID=601803&tab=DetailInfo
You do not need to use TaqMan assays. It might give u more specific assaying but it'll also be more costly since you'll have to make and purchase a probe for each mature miRNA. If you optimize your SybrGreen reaction and use high quality RNA to start you'll be fine.