I want to analyse the expression of miRNA through RT-qPCR method. I am confused with the various approaches of doing it. Should I go for designing the primer (Stem loop primer) to synthesize the cDNA and then go for the qPCR using specific primer, or I should go with the kit available for Qiagen for both RT and qPCR(using specific primer only at this step)? I want to go for multiple numbers miRNA. Your kind suggestions would be highly appreciated.
I would recommend using one of the microRNA profiling kits. I used a qPCR microarrays that profiled 96 different oncomiRs at the same time on one qPCR run. It was pretty easy and the results were great.
isolate total RNA or total miRNA with eg mirVana Paris kit, then use miRNA TaqMan assays - Applied Biosystems/Thermo Fisher has everything for qRT-PCR analysis. there are options to use RT primers specific to your miRNA sequence of interest, or "universal/random" primer to perform Reverse Transcription for all seq
There are several techniques to quantify miRNA expression but realtime qPCR based techniques are the most popular and easy. From your sample, extract the total RNA using any kit (Just make sure that it extract total RNA, which also includes miRNAs). One you get the total RNAs, you can produce cDNAs from miRNAs only. In this step you can select whether you make cDNAs from all miRNAs (using universal primers) or only from the miRNAs of your interest (using specific primers). If you want to check the access expression of more than one miRNA, I strongly recommend the universal one because you don't need to prepare individual sample for realtime qPCR for each miRNA. I use Taqman Advanced miRNA cDNA synthesis kit from ThermoFisher to produce cDNA from miRNAs using universal primers. Once you have cDNA, you can proceed for real-time. If your miRNAs are less (more or less 10) and you need to replicate the same experiment a lot of times, I recommend to order single tubes (Taqman Advanced miRNA Assays) for each miRNA and mastermix from ThermoFisher. Once you have all these, you can do the realtime PCR in your own plate setup. However, in the thermoFisher website, you can design the 96 well plate and order a minimum number of plates. Also, if you have a lot of miRNA targets or you don't know the expression profile of the miRNAs in your sample, I recommend you to use 384 well plate. Note that you have the option to design the plates too.
I have not used other products, but I believe they basically use the same principles.
Hope this will help. Good luck.
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