Anchored oligo dT and Thermostable reverse transcriptase - temperature compatibility between RT and oligo dT priming (Feb/25/2008 )

Hello Experts,

I am setting up a RT-PCR reaction using "Anchored Oligo dT" as primer and using thermostable reverse transcriptase as the polymerase.

I am concerned that the temperature optimum of working of the reverse transcriptase might be too high for the dT oligos.

Anyone with experience in doing RT using oligo dT, can shed some light on this issue, please!

thanks.
brami

Why would you think so? The dT sequence is quite long typically (20 bp or more), and the DNA:RNA binding is tighter than the DNA:DNA binding, so the Tm will be higher. RT reactions are typically done at 50 C, so I see no particular problem.

Is 50C enough to remove RNA secondary structure (if I need to get full length c-DNA copies)?

If I need to perform RT at higher temperatures, annealing could be a problem since the typical Tm's of oligo dT is in the 42-45C range.

Or am I missing something very obvious?!

thanks.
brami

I'm not up on the latest available RT enzymes, but last I checked it was difficult to find ones which worked above 50C, which was a major difficulty in solving the secondary structure problem. Of course, once the enzyme starts, the Tm becomes largely irrelevant for extension. The standard dT anchored oligo is TTTTTTTTTTTTTTTTTTTTVN where V = anything except T, N = any base. You can just send this sequence to the synthesis companies and have it made. You can add 5' bases to this to allow PCR priming of the amplified strands using better primers.

Invitrogen Thermoscript reverse transcriptase (RNase H activity reduced) works between 50-65C.
Thanks for your useful replies !

Thermoscript in my hands wasn't too good for longer parts. Transcriptor (Roche) and SuperscriptIII (Invitrogen) worked fine for me at 55°C.

Other than that, if your secondary structure is really bad: is there a possibility to pre-denature your RNA for 5-10 minutes at 65-70°C and then proceed to reverse transcripton?

You can also stabilize the RT at higher temperatures by adding trehalose. This allows you to run RT at temperatures as high as 60°C. It's what they use to make the full-length cDNA libraries at RIKEN. Very good.

Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA. Carninci* et al. PNAS Vol. 95, Issue 2, 520-524, January 20, 1998

Ginger

Thanks "gingerspice" and "varius".
I incidentally got the same reference from another source.
People like you make this discussion forum so informative and great to be a part of!

Brami

Hello ginger spice,

And what reverse transcriptase do they use along with Trehalose?????

thanks
brami
_______________________________________________________________________