Northern Normalization

Ken Dwyer kjd at biotech.ufl.org
Wed Aug 28 14:53:46 EST 1996


Susan Patrick wrote:
> 
> I was hoping to use an internal standard, a so-called constituitively
> expressed gene, to normalize my northerns.  I have read some about how
> much these genes may vary in some cells, during differentiation, etc.
> Any suggestions on how I should go about insuring loading consistent
> amounts of RNA?  (I am working with differentiating F9 (murine) cells.)
> 
Susan,

When ever I need to normalize northerns I use a housekeeping gene that
does not undergo developmental regulation,  I normally use actin or
tubulin.  If both of these are regulated in some manner, I probe with
oligo dT(18)-P32.  Briefly, I first hybridize with all experimental probes
and scan the autorads with a densitometer.  Last,after stripping the
membrane I probe with p32-oligo dT(18) labeled at the 5' end with T4
polynucleotide kinase.  The blots are hybridized in 2XSSC,5% SDS, 2X
Denhart's Solution, and 1 ug/ml carrier DNA with 10(6) cpm
P32-oligo-dT(18) overnight at 21c.  The oligo-dT northerns are quantified
with the  densitometer.  The autorad is imaged from the 9.5 kB RNA
standard to the 0.24 kB RNA standard and the mean area of oligo-dT(18)
hybridization computed.  I then compute the ratio for experimental /oligo
dT(18).

Good luck

Ken Dwyer



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