I'm trying to generate full-length cDNA of my gene of interest (7kb) but have trouble doing so. I was wondering if maybe there are certain RNA extraction methods that are better suitable for this purpose than others.Currently I'm using the RiboPure kit to extract RNA from my lymphoblastoid cell lines.
Dear Lena,
Your currently method is enough for a high quality RNA extraction (Chek the RIN of your RNA before cDNA synthesis in Bioanalyzer). Possibly, the problem is in your method for cDNA synthesis from Total RNA. You should chek your specific primer for full-length cDNA synthesis. Other option is carry out the cDNA synthesis with oligoDT18 and after amplify your gene with specific primers using a proof-reading polymerase.
Good Luck!
Making full length cDNA of very long mRNAs is not that easy. Using a poly-A primer, reverse transcriptase can stall , stop, or fall of after several thousand bases. In the old days, we used to piece them together from cDNAs primed with random hexamers. Today, I would just order the clone from one of many sources, such as the ESTs available. Or, make gene specific primers so that you can amplify the entire mRNA in 3-4 segments and then ligate them together. In the end, it is probably cheaper to buy the clone if available.
Using NEB ProtoScript II or Thermo SuperScript VI, I have been able to reverse transcribe at least 3 kb using a gene-specific primer. The latter product boasts over 10-kb synthesis capabilities. I have never tried that myself, but I don't doubt that it works.
I have amplified and sequenced ~3.5 kb of full length mRNA.
If you find difficulty in amplifying 7 kb, you can design 2 primer pairs and generate 2 amplicons.
Best wishes
Muhammed
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