I have been trying to amplify the human factor VIII mRNA sequence (7Kb) by reverse transcription PCR. I attempted to do this by dividing the sequence into 5 overlapping fragments similar in length. I have tried evertything from touchdown PCR to additives like DMSO and Betaine, yet 2 moths later I am still only able to amplify 2 of the five fragments. And I have tried amplifying the full length which was also unsuccessful. I think the problem could be the primers and maybe secondary structures, and problematic regions in the RNA template. Has anyone successfully reverse transcribed and PCR amplified this sequence and be willing to share primers ? Thanks
Hi,
For very long transcript it is better to use specialized reverse transcriptases which are optimized for such reactions. For example, Roche expand long RT is one such formulation for long cDNA synthesis. There are similar enzymes from few other companies. They contain specialized and optimized buffers for good RT reaction. You may also check the %GC content of your sequence and if needed use High-GC buffer for RT. Also also try to find out how much abundant is the transcript in the cells/tissue you are using. These suggestions may be useful
I'd try to do it in one reaction with a long-template polymerase. Have you checked to make sure that it doesn't have several isoforms and/or closely-related genes?
Good luck!
- Badges
- Science method