i want to know the steps for RNA reverse transcription and how i can design its primer
The RNA reverse transcription is a necessary step before PCR. As you know PCR is a technique which DNA is amplified. So if your target is RNA, you have to trasncript it to DNA. For RNA reverse transcription, you have two choices:
1. You can use the random primer [Rp] : are oligodeoxyribonucleotides (mostly hexamers) used to prepare labeled DNA from RNA, are suitable for DNA synthesis using Klenow fragments with DNA templates or for cDNA synthesis using reverse transcriptase with mRNA templates.
2. You can also use the reverse primer (that you will use in your PCR).
In conclusion:
Rp + Reverse transcriptase enzyme + RT-Buffer + dNTP + RNAsin + RNA
=====> cDNA
cDNA will be used as template for PCR.
I hope that I was clear.
Regards
Good luck.
Hello Noha,
1. There are multiple sources for understanding the steps of reverse transcription.
Fundamentally it's the synthesis of DNA from RNA using a special enzyme called reverse transcriptase. Essentially using hexameric random primers you synthesize the cDNA from a sample. Using this cDNA as a template then you assess the levels of your interested mRNA compared to the control sample.
Here you can find a simple protocol how to process your RNA to cDNA.
https://www.promega.es/~/media/files/resources/protcards/reverse%20transcription%20system%20quick%20protocol.pdf
2. For designing the rtPCR primers, you might use the "pick the primer" tool in the NCBI "BLAST" page. Ideally you design the primers flanking the exon-exon junctions, so you can avoid the genomic DNA contamination.
Another easy to use tool is universal probe library -> Assay design centre from Roche, here is the link. (if this doesn't work just google universal probe library)
https://lifescience.roche.com/shop/CategoryDisplay?catalogId=10001&tab=&identifier=Universal+Probe+Library
Hope I explained myself well enough. Please let me know if somethings are not clear.
All the best,
Krishna.
The RNA reverse transcription is a necessary step before PCR. As you know PCR is a technique which DNA is amplified. So if your target is RNA, you have to trasncript it to DNA. For RNA reverse transcription, you have two choices:
1. You can use the random primer [Rp] : are oligodeoxyribonucleotides (mostly hexamers) used to prepare labeled DNA from RNA, are suitable for DNA synthesis using Klenow fragments with DNA templates or for cDNA synthesis using reverse transcriptase with mRNA templates.
2. You can also use the reverse primer (that you will use in your PCR).
In conclusion:
Rp + Reverse transcriptase enzyme + RT-Buffer + dNTP + RNAsin + RNA
=====> cDNA
cDNA will be used as template for PCR.
I hope that I was clear.
Regards
Good luck.
Maybe what you need is a one-step RT-qPCR protocol... ;-)
http://www.bioline.com/us/mytaq-one-step-rt-pcr-kit.html
Hi Noha, the protocol extraction choice will depend to your sample.
You can test both of them and compare results (if ou have trizol and the kit of course) :)
I prefere the column spin, because protocoles are very quick and simple than trizol.
Regards
Hello,
I agree with both the researchers. I wish to add that there are several methods of RNA extraction which depend on your type of sample but I would prefer to Trizol as it is most effective method. For any reverse trancription PCR, the steps start from RNA isolation and quantification - cDNA synthesis- PCR
for cDNA synthesis,
Use the cocktail ( RNA and Oligo dT / Random hexamer - incubation - addition of Reverse transcriptase, RNase inhibitor, RT buffer, dNTP - incubation) .
for primer designing,
Please follow ,
http://www.ncbi.nlm.nih.gov/guide/howto/design-pcr-primers/
Hope it helps.
Just for primers, You can use primers which were already used /published or you can design them. It will depend to your gene target (New gene or already discover).
Regards
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