Does Reverse Transcription have to be done using PCR - (Oct/12/2005 )
Please help me!
I'm currently doing a project which requires the isolation of the g-TMT gene from Arabidopsis - Im planning to do this by isolating the total RNA, converting it into cDNA and then amplifying my specific g-TMT cDNA using desired primers, using PCR. If my lab has only the standard PCR, will I be able to carry out the reverse transcription using RTase and oligo-dTs in vitro outside the PCR? Is this possible?? If anyone knows the protocol, please help me out!
Hoping to get a response as soon as possible!
You can carry out the Reverse transcription using a primer used in your PCR if you want. If I'm not mistaken it should be the antisense primer (but as I never work with mRNa I might be wrong).
Easiest thing to do is to use a "one step RT-PCR" kit, using your normal PCR primers.
I agree with vairus
but for future reference, it was not always a one-step, one-tube deal, you can certainly just make a bunch of cDNA from total RNA
I RT my RNA into cDNA as a seperate step all the time, it is easy and goes like this, assuming EVERYTHING is nuclease-free:
250 ng total RNA template (in 10 ul tris-HCl pH 8.0)
1 ul oligo (dT)vn23 (from NEB)
9 ul sddH2O
heat at 70C 5', quick-spin, hold on ice (this will unfold any secondary structure in your RNA)
18 ul sddH2O
10ul 5X RT buffer
1 ul RT enzyme(I use the one from Progmega)
1 ul RNase inhibitor(I use the one from Invitrogen)
incubate at 42C 60'
put on ice until ready to use
proceed to PCR; nothing in the tube should interfere with downstream reactions. store at 4C. you should need only a tiny volume for a PCR. I usually make a master mix of oligodT to add to the templates, and then make a master mix of the second batch of ingredients, based on my number of samples. if you have a problem with your PCR, you can clean up the cDNA I suppose, but I have never needed to do so.
I prefer two-step RT-PCR for its versatility.
Create your cDNA libraries first from dT-oligos, then amplify the cDNA template with gene-specific primers in a normal PCR reaction.
If you want to check for the presence of another transcript later, all you have to do is amplify the cDNA library with new primers...it is not necessary for you to perform another RNA extraction/reverse-transcription.
The only concern you might have is contamination of the cDNA library.
When DNA region to be amplified is located in only one Exon (not separated by intron), UTR 5' or UTR 3' (untranslated regions) you don't need neither to extarct your RNA nore to reverse transcribe it ! if this is the case, you can to obtain your sequence from a genomic DNA by a classic PCR and subsequently sub-clone it.
But, if you are planning to do an expression experiment and use cDNA probe, you have to reverse transcribe your RNA.
Am I right ?
yes, I think so, I done it for my genes and everything is OK !
I agree with aimikins I am using that protocol and it is very easy!!
oh, hey, another quick point...
I find that it is a good thing to make your cDNA from your RNA as quickly as possible. No matter how careful you are, DNA is still more stable than RNA. So, cook up your cDNA before storage and it will last you a long time