I am doing PCR for microRNA, I isolated total RNA, then using stem loop primer, I make cDNA, and now doing PCR with bioRad cyber green kit. Some of my microRNA sample have very good melting curve and cq value. But others are very bad. Can anybody explain? Is total RNA work for microRNA processing without purification, is total cDNA synthesis kit work for microRNA, is normal PCR ingredients work for microRNA PCR.
Any suggestion please.
1.Total RNA can be used for quantifying miRNA without any problem. Although self designed primers work efficiently, you need to standardize them thoroughly. To avoid such troubles. I would advise you to use Applied Biosystems reagents (miRNA RT kit, specific miRNA RT and taqman primers). I have never faced any such problem if handled properly, even at 20ng total RNA (Can be used for up to 1 ng).
2. I have used total cDNA kit (Applied Biosys) with specific miRNA primers, excluding random hexamers, without any problem. They do work efficiently. I have no Idea about normal PCR ingredients. It may work, depending on the efficacy RT enzyme in appropriate buffer.
Total RNA can be used if prepared by a method (e.g. miRVana miRNA isolation kit) which preserves the small RNAs. Trizol loses many miRNAs and would not yield reliable results.
If miRNA-specific TaqMan probes are not used in your experiments, highly similar miRNAs may be amplified by a single pair of primers. Check the Applied Biosystems website.
Hope this helps.
the debri is causing bad pcr results
there are polymers that yield good amounts of very pur micro rna s from exosomes
pure exo is the best one I have heard of
http://www.gentaurpromo.com/exosome_kits/
I am using Trizol Reagent for total RNA extraction and it works good for microRNA quantification. what do you mean by bad melting curves? do you get extra peaks with different Tms? or it is not as sharp as you expected? it could be inhibitor carry over from the RNA extraction step, try a serial dilution of your input RNA in cDNA synthesis step. check your NO-RT control if you found similarity between its melting curve and your samples try to enhance DNase treatment step. by the way, Stem-loop primers sometimes cause problem in samples with low template concentration. set up several cDNA synthesis reaction with different concentration (mostly lower concentration) of primer input .
good luck!
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