smear Northern

James Tsuruta jtsuruta at med.unc.edu
Tue Aug 20 21:26:06 EST 1996


Hi,

You say that you are having trouble detecting a rare transcript by
Northern analysis and I think that you also mentioned that the rRNA bands
were were fine (in reference to the conclusion that your RNA is not
degraded).  This leads me to think that you are using total RNA in your
Northern analysis.  My suggestions are:

1)  use poly A+ RNA (10-20 ug/lane)

2)  use downward alkaline blotting (reported to give higher signals), try to
    keep your gel thin for highest transfer efficiency (0.5cm)

3)  make the highest specific activity probe that you can
    (I personally dislike riboprobes since they are so hard, in my hands,
    to strip off of blots, but if you are only going to use this blot once
    that may be the way to go:  you could use more than one hot nucleotide
    and use probes to multiple regions of your message) or you could use 
    assymetric PCR and synthesize a single stranded anti-sense probe with 
    more than one labelled nucleotide.

4)  Some membranes give better results than others, I personally like
    MagnaGraph from MSI but have never done a side-by-side comparison to 
    show that this membrane gives a higher signal than others.

5)  Pre-flash your film (see Manniatis)  this is especially important when
    signals are low _and_ essential if you are quantitating levels since
    the response of film is _not_ linear at low exposure levels.  (As I
    understand it, a silver grain needs to be hit multiple times before it 
    is actually "exposed" thus film actually has a sigmoidal exposure curve)

6)  Be sure that your UV-crosslinking is optimized.  If you have a
    Stratalinker-type device you are probably close to optimal
    but if you have to use a UV-light box like I do....be sure that someone
    has done a dose-response expt to "calibrate" the correct time for 
    optimal x-linking. (I used a dot-blot with constant amts of RNA and varied
    the exposure to 310nm UV, probed this with a labeled cDNA and determined
    the optimal x-linking time, this will vary depending upon the age of your
    UV lamp and the age of the UV-filter)

7)  You can get around (5) if you use a PhosphorImager since the response
    of the phosphoscreens is reported to be linear

8)  Be sure to use an enhancer screen and expose @ -70C.  Someone told me
    that the highest signals are obtained if you do this:
    screen#1:film (pre-flashed side towards screen#1):blot:screen#2
    
    Can anyone tell us why screen#2 is used?

9)  I also remember that signal to noise ratios can be increased by
    clearing the emulsion off of a certain side of your film using bleach
    Unfortunately I can't remember which side, and I recall that the actual
    signal is diminished significantly but it sure did make pretty films
    when done correctly.

10) Sorry if you knew all of this or if it has already been posted (I'm
    having trouble with my newsreader and can't tell what's been posted in
    response to your query!

    If I've made any incorrect explanations/assertions or if anyone has
    other hints....Please post!!!!

James Tsuruta, PhD
The Laboratories for Reproductive Biology
University of North Carolina at Chapel Hill



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