Hello everybody.
I aim to perform a Ribonuclease Protection Assay (RPA) with a specific RNA target that I will transcribe (MegaShortScript kit). I would like to know some methods (fast and low-priced) for validation of this step before going to the RPA experiment.
Thank you in advance.
Idriss
You may try to harvest and purify total RNA and do reverse transcription to obtain cDNA. Then design primer to flank the transcript you target.
Remember using accession number for transcript but not genome.
Thank you very Much! I hadn't even thought of that.
As my PCR product (target transcript) is very short (23nt, ), do I need to purify them or I can directly perform the RPA (hybridization, digestion etc.).
Thank you in advance
23nt is too short to do PCR. I suggest you to do northern blot.
You may try to read this article.
Northern Blots for Small RNAs and MicroRNAs
http://cshprotocols.cshlp.org/content/2014/7/pdb.prot080838
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