Problems with RT-PCR

alex dobrovic adobrovi at medicine.adelaide.edu.au
Thu Jun 25 21:13:19 EST 1998


Tubulin, like actin and GAPDH has multiple pseudogenes and if your DNAse
treatment is not 100% efficient (and it rarely is) will amplify up from
residual DNA. You need to choose a universally expressed low level message
like abl or B2-microglobulin as a control.

 >I am interested in doing semi quantitative RT-PCR on targets that have
>very low expression levels. To standardise all my reactions I have been
>amplifying tubulin (T26 variant) in the same reaction tube as the
>target. In the past, I have varied the cycle number from 20-40 cycles
>with the T26 plateauing out at about 32 cycles, whereas, the target
>starts to appear around 30 cycles.  This worked the first couple of
>times about a month ago but since then I have failed to get any target
>bands.  I am even now having difficulty in getting bands when no T26 is
>amplified in the same tube.  I am DNAse1 treating my samples
>,conditions, 7ug RNA, 3ul 10x PCR buffer, 3ul 25mM MgCl2, 40u Rnasin,
>7.5u DNAse1 in a final vol of 30ul, incubate 37degs for 15min then heat
>inactivate at 65degs for 10min.  I use 5ul of this to make first strand
>cDNA using the Superscript kit ( Life technologies).  I do not use
>Rnasin during this step and I also reduce the amount of buffer/Mg added
>to the sample since the RNA sample already contains salt ( but still
>give the correct final concentration)  I then use 2ul and 0.4ul for
>PCR.  I have been worrying that I have got RNase contamination but the
>tubulin always works well.  I have since replaced everything and it's
>still not working.  The only thing i can think of, is that I'm getting
>very low level of RNA degradation somewhere along the line which is
>sufficient to degrade the target RNA but still leave plenty of T26 RNA
>which is much more abundant.  Is it better to use primer sets that span
>exon/intron boundaries and so remove the need for DNAse digestion?  I am
>at a loss, so any help will be very appreciated.
>
>Thanks Gary


Alexander Dobrovic, Ph.D.
Chief Medical Scientist
Department of Haematology-Oncology
The Queen Elizabeth Hospital
Woodville, SA 5011, Australia

Affiliate Senior Lecturer
Department of Medicine
University of Adelaide

Tel 61-8-8222 7676 (new as from April 1998)
Fax 61-8-8222 6046
adobrovi at medicine.adelaide.edu.au





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