Whether Random N6 primer will miss the first several nucleotides from the 3' of the template RNA?
Ali Mahmoudpour is absolutely right. I would just ask why you are concerned about the final three nucleotides of the RNA? More background could help to better answer the question. Is it an mRNA? If so it is likely to be polyadenylated in which case you can capture the entire mRNA sequence by reverse transcription from an oligo-dT primer. Also if you are concerned about capturing the extreme 3' sequence of the RNA then a method called 3'-RACE (Rapid Amplification of cDNA Ends) can be used to capture the 3' end sequence. ThermoFisher even has a kit for it: https://www.thermofisher.com/ca/en/home/references/protocols/nucleic-acid-amplification-and-expression-profiling/cdna-protocol/3-race-system-for-rapid-amplification-of-cdna-ends.html
Ali Mahmoudpour Zachery R Belak : Thanks very much. In other words: the product will be a little shorter or longer than the template RNA, am I correct ?
More details: My template is not mRNA (without polyA tail) and I focused on the first nucleotide in the 3' end (which is added artificially). Is there any methods or techniques to investigate the first nucleotide for these unkown RNA sequences (more then ten thousands).
Chang-chang Cao the best option is to use T4 RNA ligase to attach a DNA adapter oligonucleotide to the 3' end of your RNA. Then do reverse transcription using the reverse complementary primer to the adapter primer. Finally, do high-fidelity PCR using the adapter primer and a primer specific to your RNA sequence. You can send the resulting PCR fragment directly for sequencing using the adapter primer and this will tell you the identity of the 3' nucleotides. I have attached a diagram and a link to a protocol for attaching DNA oligo adapters with T4 RNA ligase.
https://international.neb.com/protocols/2018/10/17/protocol-ligation-of-an-oligo-to-the-3-end-of-rna-using-t4-rna-ligase-1m0204
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