Primers for RT reaction - (Jul/11/2005 )
Hi,
I would like to know if oligo-dT are a good primer use while performing RT reaction.
Thanks
It's a terrible primer, but it has the huge advantage of binding to every poly-A tail of eukaryotic mRNA. You would never choose it for any other reason. I like the version with a 3' A, G, or C.
Why is that?
The all-T version spells "strand slip" all over it. Of course, you may not care about consistency in the length of your product. If, however, you use the primer for PCR as well as mRNA -> cDNA conversion, you will end up with a variety of PCR products of differing lengths. These are fine as cloning fragments, where a single strand is cloned and selected for sequencing, but useless for direct sequencing from the PCR product (at least from that end).
The 3' base binds to a specific location on the mRNA or cDNA, and also tends to pin down the 3' end of the primer. Since extension only happens when the 3' end is annealed, the odd base allows extension at higher temperatures. Some people use primers of the form TTTTTTTTTTTTTTTT {A, G, C} {A, G, C, T} for similar reasons.
Cool!
Thanks for the info phage434.
Ohhh, what are the main disadvantages of oligoT?
The main disadvantage of oligo dT is the 3' bias that it creates. By this I mean that everything at the end of your transcript is going to have a higher representation than the start. This is particularly evident when using RNA of somewhat questionable quality and if the desired products are large or have considerable secondary structure.
Hope this helps.
Scott