RT-PCR question

[douglas l feinstein]

Subject: RT-PCR question
From: Adi Santoso, santoso at badlands.NoDak.edu
Date: Sun, 2 Oct 1994 17:54:25 GMT
In article <Cx24Ep.4r5 at ns1.nodak.edu> Adi Santoso,
santoso at badlands.NoDak.edu writes:
>i try several time RT-PCR with 3' primer using oligo dT (16 mers) just 
>never work. I check all the reagents and template RNA, they are all 
>oke...!!! any suggestion for me.  I just almost give up with it..
>
>ADI
>Dept. of Biochemistry
>North Dakota State University

Subject: Re: RT-PCR question
From: Sean David Moore, smoore at mail1.sas.upenn.edu
Date: 4 Oct 1994 16:52:57 GMT
In article <36s199$647 at netnews.upenn.edu> Sean David Moore,
smoore at mail1.sas.upenn.edu writes:
>If all of the reagents are "ok", then the reaction will work..unless the 
>target isnt there.   
>
>
>
>Adi Santoso (santoso at badlands.NoDak.edu) wrote:
>: i try several time RT-PCR with 3' primer using oligo dT (16 mers) just 
>: never work. I check all the reagents and template RNA, they are all 
>: oke...!!! any suggestion for me.  I just almost give up with it..
>
>: ADI
>: Dept. of Biochemistry
>: North Dakota State University
>
>--
>Sean Moore
>
>VAMC Philadelphia,
>Medical College of Pennsylvania,
>and the University of Pennsylvania...
>
>.
>
>smoore at sas.upenn.edu
>
>

Subject: Re: RT-PCR question
From: Gerri Newfry, gnew at orion.it.luc.edu
Date: 4 Oct 1994 16:41:06 GMT
In article <36s0j2$n0d at apollo.it.luc.edu> Gerri Newfry,
gnew at orion.it.luc.edu writes:
>
>I use random primers for the RT step, which are supposed to (according
to 
>clontech) 
>work better.  Also, I started using a cDNA cycle kit from Invitrogen.
>
>Good luck,
>
>Gerri
>
> Adi Santoso 
>(santoso at badlands.NoDak.edu) wrote:
>: i try several time RT-PCR with 3' primer using oligo dT (16 mers) just 
>: never work. I check all the reagents and template RNA, they are all 
>: oke...!!! any suggestion for me.  I just almost give up with it..
>
>: ADI
>: Dept. of Biochemistry
>: North Dakota State University
>
>--
><>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*<>*
>
>		gerri...gnew at orion.it.luc.edu				   
>

Subject: Re: RT-PCR question
From: Richard R. Hardy, hardy at mighty.fccc.edu
Date: Tue, 04 Oct 1994 14:53:32 -0500
In article <hardy-0410941453320001 at mighty.facs.fccc.edu> Richard R.
Hardy, hardy at mighty.fccc.edu writes:
>> Adi Santoso (santoso at badlands.NoDak.edu) wrote:
>> : i try several time RT-PCR with 3' primer using oligo dT (16 mers)
just 
>> : never work. I check all the reagents and template RNA, they are all 
>> : oke...!!! any suggestion for me.  I just almost give up with it..
>> 
>
>Try doing your RT reaction with random hexamers and then the PCR with a
>specific 3' primer.  For big transcripts, we sometimes get low signals
>with oligo dT, even doing the PCR with specific 5' and 3' primers (when
>they're located 5' in the gene).  Even if Oligo-dT works well for the RT,
>it still may not be a very good primer for the PCR (GC content of 0!)...
>
>-- 
>R. Hardy
>Member, Institute for Cancer Research,
>Fox Chase Cancer Center, Philadelphia, PA
>(215) 728-2463

Subject: Re: RT-PCR question
From: Annette C. Hollmann, ah690549 at mbcr.bcm.tmc.edu
Date: 4 Oct 1994 22:01:06 GMT
In article <36sjb2$7k4 at gazette.bcm.tmc.edu> Annette C. Hollmann,
ah690549 at mbcr.bcm.tmc.edu writes:
>In article <Cx24Ep.4r5 at ns1.nodak.edu> santoso at badlands.NoDak.edu (Adi
Santoso) writes:
>>i try several time RT-PCR with 3' primer using oligo dT (16 mers) just 
>>never work. I check all the reagents and template RNA, they are all 
>>oke...!!! any suggestion for me.  I just almost give up with it..
>>
>>ADI
>
>I had the same problem, using the Perkin-Elmer GeneAmp kit.
>I ended up switching to random hexamers for the RT step.
>Perkin-Elmer techies have suggested that anchoring the dT tail with the
>last base of the gene might help.
>
>They also suggested that I make sure that I do the initial 10 minute
>incubation at 20 to 25 C, and that I increase the RT time from 15 min to
>60 min.
>
>If you are using the Perkin-Elmer kit and have no particular reason to
>use the dT primer, I would suggest switching to random hexamers.
>
>Annette
>ah690549 at mbcr.bcm.tmc.edu
>
>Good Luck
>
>

Subject: Re: RT-PCR question
From: Tracy Aquilla, aquilla at salus.med.uvm.edu
Date: Tue, 4 Oct 1994 23:44:57 GMT
In article <aquilla.1131701937L at sadye.emba.uvm.edu> Tracy Aquilla,
aquilla at salus.med.uvm.edu writes:
>In Article <36s199$647 at netnews.upenn.edu>, smoore at mail1.sas.upenn.edu
(Sean
>David Moore) wrote:
>>Adi Santoso (santoso at badlands.NoDak.edu) wrote:
>>: i try several time RT-PCR with 3' primer using oligo dT (16 mers)
just 
>>: never work. I check all the reagents and template RNA, they are all 
>>: oke...!!! any suggestion for me.  I just almost give up with it..
>>
>...(deleted)...
>>If all of the reagents are "ok", then the reaction will work..unless
the 
>>target isnt there.   
>>
>
>Not if the wrong PCR profile is used. If you tell us more about the
>parameters of your experiment, maybe someone can help. I think a 16mer of
>oligo dT has a pretty low Tm (about 32C at 50 mM Na conc?). I suggest
using
>a longer primer, and one that's specific for your gene, instead of the
dT oligo.
>
>
>
>Tracy Aquilla, Ph.D.
>Molecular Physiology and Biophysics
>University of Vermont
>aquilla at salus.med.uvm.edu


Actually, we've using random primers, and we typically mix RNA (1 ug)
with  300 ng random hexamers, heat to 65 for 5 min, then bring temp down
to 37. Then we wait about 1-2 min, and add the reaction mix containing
enzyme. At end (after 1 hr), sample is heated 5 min at 95oC, then brought
to Room Temp. We spin briefly, add 80 uL H20, and do PCR on a 5 uL
aliquot (total volume at end is 100 uL) using specific primers. This
always !!! works. But, the yield may be low since the annealing of
hexamers never goes below 37oC. I am going to try to modify by bringing
temp down to R.T. after 65oC, and then adding RT enzyme, and slowly
bringing temp up to 37oC (using a slow ramp setting on the machine) then
do 37 for 1 hr. 
			Might be interesting!

dlf