Reverse Transcription with oligo dT or hexamer primers? - What do you prefer and why? (Apr/20/2006 )

Hi,

so there are adventages and disadventages of both methods. The aim of this threat is to
give beginners (like me) a guide when to use which primer and which dilution to use afterwards for your real time. It would be also nice to know if there are housekeeping genes as reference, which are better used with random primers or whith oligo dT. Starting point is your purified and (hopefully) good quality T-RNA.

I'll give a start:

random primers: good if your primer pair was designed more at the start of the transkript, so that there is 3 - 8 kBp until the end of the transcript. disadventage is, that also ribosomal RNA will be amplified, leading in some cases to a lower template concentration.

oligo dt: we're using superscript II from Invitrogen and in most cases they work pretty well.

Generally, I prefer oligo dT primer over random haxmer because its specificity toward RNAs with poly A tail. A disadvantage is that reverse transcription may not reach the 5'-end of long mRNAs.

Sometimes, random primer is necessary for RT such as for the detection of non-coding RNAs.

Random hexamer because I specifically deal with bacteria, not eukaryotes.

-Matt