We have isolated miRNA / Total RNA using Total nucleic acid isolation kit from Life Technologies. The quality of RNA is good. But the quantity is low (10-100ng). I see that Qiagen has three step reaction. 1 miscript Reverse transcrion kit. 2.miScript preamp PCR ( applification of cDNA. 3. Sybr green (interested miRNA forward and universal reverse primers).
However, these kits are very expensive considering that I have higher number of samples.
I would like to know suggestions for alternatives?
Hello,
I have been using this kit for transcription The High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with really low RNA concentrations. Add maximal volume of RNA and even more. I have been adding 20 microliters of RNA with 2,2 microliters RT buffer, 2,2 microliters Random Primers , 1 microliter dNTP Mix, 1 microliter RNase Inhibitor and 1,2 microliter MultiScribe™ Reverse Transcriptase it is all included in kit (Rnase included type of kit).
Hope this will help you. Best regards, Bojana Mohar V.
we typically follow Applied Biosystems qRT-PCR protocols, with miRNA or mRNA TaqMan assays or sometimes Sybr. works well for FFPE-derived RNA (and low NA yields and sometimes low quality have to be expected for these challenging samples, esp old blocks)
Thanks for the replies.Thanks so much for taking time to answer my questions.
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