RNA Slot-blotting woes

Patrick HJ Falckh p.falckh at rmit.edu.au
Mon Jun 10 18:04:34 EST 1996

'Lo Gabriel,

  The problem with trying to use the same amount of RNA on a Northern 
compared to a Slot-blot is the amount of RNA binding per square mm of 
membrane; remember that the amount of RNA applied in a Northern will 
travel some distance (say 10-15 cm) and this separation essentially 
'dilutes the amount of RNA being applied at a single point.  Hence 
loading 20ug total RNA on a Northern may only represent 1/100th of the 
band you see when you probe it.  In addition, the binding capacity of 
the membrane must play an important role.  In slot-blot analysis' I've 
used a serial dilution of total RNA in the range 0.025-2 ug.  
  In addition I've applied 2 concentrations of a 'standard' (total RNA 
from a single extraction of a tissue that gives good binding of my 
probe) at opposite corners of my slot-blot.  This 'standard' allows me 
to standardise the results from different blots just in case there is 
some slight difference in the treatment of the membranes.

As commented by Jim Meador use of Northerns of Slot-blots is not the 
best method for quantitation and the RNase protection assay is much 
better.  With the increased quantitativeness of the RNase assay comes 
the more tedious and costly expense, although the results are 
more-often-than-not worth it.

   Patrick HJ Falckh PhD                                 #     #     
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