I have a 27 nt siRNA that is cleaved by DICER enzyme into a smaller 21 nt fragment. Due to physical and chemical modifications, this 27 nt will be cleaved on an exact point and will originate a single 21 nt product (all other possible 21 nt variations are not produced due this modifications). All this information has already been published by other authors.
I submitted a manuscript and one of the referees requested to prove that, in my experimental model (cell culture), the 27 nt siRNA is actually been diced into the predicted 21 nt. The most straightforward assay to confirm this would be a northern blot. However, I don’t have structure to do this assay at my lab (radioisotopes need special handling conditions and authorizations that would take weeks to set up).
The referee suggested that I could answer my question by a qPCR. I checked the stem-loop RT-qPCR and it would not function, as the 27 nt and 21 nt has the same sequence at 3’ end. I also studied a few other protocols and I end up with the same problem. Both sequences (27 nt and 21 nt) are identical except for the additional 6 nt at 5’ end of 27 nt siRNA.
So, my question is, is there anyway to answer my question by qPCR (is the 27 nt siRNA being diced into the 21 nt predicted siRNA?)?
Sequence examples:
27nt 5’ – YYYYYYXXXXXXXXXXXXXXXXXXXXX – 3’
21 nt 5’ – XXXXXXXXXXXXXXXXXXXXX – 3’
If I understand this correctly the products are way too small for qRT-PCR investigation.
Perhaps you can detect by northern with non-radioactive probe? 5' modification of the 21 nt could be done for fluorescence, or even biotin, which could be used to probe well separated products. You might want to synthesize the 27 nt and 21 nt as calibrates for your gel system. A quick google search suggests a potential starting point:
Olá Bruno!
Is there a larger precursor at the 5' end you need to worry about?
You can amplify both small RNAs using the poly-A tailing method plus a forward primer that hybridises with both of them or that would just hybridise with the 27nt fragment. I suppose if you run this in semi-quantitative conditions (or even qPCR) you should be able to see a change in the relative abundance of the 27nt fragment to the common 21nt sequence. Success will greatly depend on the sequence of the YYYYY's.
Can email you some protocols if needed.
Maybe if you use an RNA Ligase and do a time limited ligation - the new product formed after RNA ligation from the 21 vs 27 nt fragments will amplify the difference of 5-6 nucleotide: After multimeric ligation where the difference will mathematically add up and be easily distinguishable on a PAGE gel.
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