polyadenylation and RT-PCR - (Dec/01/2008 )

Hi,

I have been trying to reverse transcribe total bacterial RNA by polyadenylation and oligod(T) primers... I used the polyA polymerase kit from Epicentre, cleaned up the reaction on spin columns and did the RT with both Ambion and Invitrogen kits... afterwards I did a PCR for some genes and all I get is a smear near the wells, while I don't have any problem when I do the RT with gene specific primers... I ran the RNA on the gel after poly(A) reactions and the rRNA bands show lower migration, their size is increased, so I guess the polyA reaction is not the problem...


Any suggestions?

thanks!

Not all prokaryotes use polyA tails, so this is probably why your RT with specific primers work but your polyA does not. You'd be better off using random hexamer 'primers' instead. Also, I seem to recall that polyA tails in prokaryotes will target transcripts for degradation...See these papers

Régnier P, Arraiano CM (March 2000). "Degradation of mRNA in bacteria: Emergence of ubiquitous features". Bioessays 22 (3): 235–44. doi:10.1002/(SICI)1521-1878(200003)22:3<235::AID-BIES5>3.0.CO;2-2. PMID 10684583.

Steege DA (August 2000). "Emerging features of mRNA decay in bacteria". RNA 6 (8): 1079–90. PMID 10943888. PMC: 1369983, http://www.rnajournal.org/cgi/pmidlookup?v...p;pmid=10943888.

Slomovic S, Portnoy V, Liveanu V, Schuster G (2006). "RNA polyadenylation in prokaryotes and organelles; different tails tell different tales". Critical Reviews in Plant Sciences 25 (1): 65–77. doi:10.1080/07352680500391337.

Yeah, I know that not all of them has the polyA tails... This is why I used the polyA polymerase to add a polyA tail, but somehow it doesn't work as I want it to...