choice of reverse transcriptase - (Apr/11/2006 )

Hi,
i am planning to do a rt pcr in tuberculosis , kindly tell me what is the criteria for selecting the reverse transcriptase? through articles, i saw both Mulv and amv is used, how to make a decision, which one will be suitable for mRNA level detection of infectious diseases?
Thank you in advance

-niveda-

QUOTE (niveda @ Apr 12 2006, 03:37 PM)

Hi

In my humble opnion, that depends on what you want to do? to construct a cDNA libaray, or just detection of infectious diseases? If for the former purpose, you'd better choose the MMLV, if for the latter, AMV is enough!

-Cinba-

Hi,

I think that even for constructing cDNA library, AMV is more viable. One thing that is important in constructing the cDNA library is to obtain full length cDNA. RNA transcripts contain many secondary structures which can be denatured through heating. The optimum temperature for MMLV is about 37C
whereas for AMV, it can reach up to 45C. Thus, as AMV is more thermostable, the chances of getting a full length cDNA will be higher.

Although AMV might from secondary hairpin loop, this can be countered by addition of sodium pyrophosphate. U might refer to Promega's Universal RiboClone cDNA Synthesis System in which AMV is used.

Hope that this helps.

Regards,
Sang Ging

-sangging-

thank you for your prompt reply

-niveda-

I would try MMLV RT from Ambion or Invitrogen.

For your purposes, it probably will not matter.

-Matt

-MisticMatt-