Will isolate RNA from tissue. I will not be running RT-qPCR, but Reverse Transcription PCR. For qPCR typically RNA is used instead of mRNA, I wonder if the same applies to RT-PCR. I only want to make cDNA and then eventually amplify a specific sequence from double stranded DNA. I won't be quantifying any gene expression...
You are welcome. If you have enough RNA it's a good option to try the best primer for your gene of interest. In the end it depends on RNA stability and also secundary structure. In my case I used Maxima Reverse Transcriptase from Thermo Fisher that can RT up to 20kb and it worked very well. I've seen some protocols which people use RNAseH after 1st strand synthesis but I didn't have to.
Good luck with your protocol!
Hi Antonios,
You can proceed the same way as you would for qPCR. You can make your RT using oligodT, random primer (or both which I prefer), or with you specific reverse primer. Just be sure that your RT enzyme works with the size of your sequence of interest in case it's too long.
Thanks a lot, Andre, I have heard before that OligodTs and random primers mixed are a good combination indeed. I also was almost convinced that I can proceed with total RNA, otherwise I would risk skewing result or have even more sources of possible error/contamination by further purifying... I am doing this experiment for the first time, so I am thinking this might be a good opportunity to evaluate which primer selection gives me a better yield so maybe Ill run a reaction with Oligos, one with randoms, one with both, and maybe one with specific reverse primer. I am slightly hesitant to use reverse primer though as I'd rather have a more intact cDNA and then amplify my sequence of interest. I have a positive control reaction where my control RNA and control primers are supposed to make the same size cDNA as my sequence of interest, very good to mention about RT enzyme capability!! Thanks a lot once again!!!
You are welcome. If you have enough RNA it's a good option to try the best primer for your gene of interest. In the end it depends on RNA stability and also secundary structure. In my case I used Maxima Reverse Transcriptase from Thermo Fisher that can RT up to 20kb and it worked very well. I've seen some protocols which people use RNAseH after 1st strand synthesis but I didn't have to.
Good luck with your protocol!
If you want to go over the way to Show a "replication" of any Agent mRNA is necessary. For all other cases total RNA, e.g. extracted by Trizol methode, is completely sufficient for any conventional RT-PCR. The best way would be to choose an amplicon between 250 and 400 bp. In your case I would take the specific primers from the start but you also can use the "uspecific" primers as well.
I agree with the above: For nearly every application total RNA is definitely sufficient
What is more isolation of mRNA from total RNA can sometimes lead to actual loss of mRNA and as a consequence reduces the efficiency of your RT rather than increases it
RT from total RNA using random hexamers with Oligo dT is straightforward and reliable
Trizol method it is!
Ran my practice/control experiment and waiting for sample to come in!
Now fingers crossed for RNase free environment throughout!
(I ended up choosing only OligodTs for my control experiment thus far, but when I use sample I will have three separate reactions one with oligodT, one with hexameters, and one with both. I really want to see which will give me better quality, if any difference in the end of the day)
My amplicon in both control and sample is 500 base pairs. Control worked so I am confident about going forward once I have sample. I used the Superscript II RT system.
Thanks a lot everyone!!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA