High background on Northern Blot

Tracy Aquilla aquilla at salus.med.uvm.edu
Thu Sep 22 16:18:37 EST 1994


In Article <94264.201910GUT at QUCDN.QueensU.CA>, <GUT at QUCDN.QueensU.CA> wrote:
>Dear netters,
>
>I wish to get some advice from any one with experience in performing Northern h
>ybridization. Briefly, I transferred total RNA onto the Nytran nylon membrane a
>nd probed the mRNA with a fluorescein-dUTP-labeled plasmid(pUC19 plus a 1.1 kb
>insert). The hybridization protocol followed the instruction manual from Dupont
>, supplier of the nonradioactive nucleic acid detection kit. Essentially, the
>hybridization buffer contains 0.25 M sodium phosphate (pH 7.2), 5% SDS, 1 mM ED
>TA and 0.5% blocking reagent supplied by Dupont. The detection system seems to
>be a much better alternative than the radioactive approach. A clear signal was
>seen after 5 min exposure of the film. However, the background of the autoradio
>graph was very high. I seems that my plasmid probe also crosshybridized to the
>28S ribosomal RNA. Several factors may be responsible for the prblem. One possi
>bility is that I used the whole plasmid as a template in probe labelling. I als
>o heard that nylon membrane usually generates more noise than nitrocellulose
>membrane. Finally, I am wondering if I should isolate poly A mRNA to avoid the
>nonspecific hybridization problem. Any suggestion will be greatly appreciated.
>
>T. Andrew Gu
>Biology Department, queen's University, Kingston, Ontario, CANADA
>e-mail: gut at qucdn.queensu.ca
>

    Did you pre-hybridize in the blocking buffer before adding the probe?
This should help reduce background. When doing chemiluminescent detection,
it is *very* important not to use too much chemiluminescent substrate, and
to rinse it all off carefully before exposing to film, otherwise the
background will be very high. If the background is still high, you may be
able to reduce the exposure time. Making your probe more specific (i.e.
eliminating vector sequence) should also help to minimize non-specific
hybridization. Charged nylon membranes can give high background signals
(after  all, they're charged, and will bind charged compounds
non-specifically), particularly if they aren't handled properly. Blocking is
often critical with some nylon membranes, but they generally need more
thorough washing too. In my experience, charged nylon membranes are far
superior to nitrocellulose, particularly if you want to strip and re-use the
blot several times. If the bands you are interested in do not migrate close
to the rRNA bands, purifying mRNA is not really necessary.

Tracy Aquilla, Ph.D.
Molecular Physiology and Biophysics
University of Vermont
aquilla at salus.med.uvm.edu



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