cross-hybridization in T7 RNA assay

[ryan at mbcf.stjude.org]

In article <17031 at news.duke.edu>, rla at canctr.mc.duke.edu writes:
> We are  picking up a lot of anti-sense msg in vivo and in reconstruction
> expts using T7-driven hot RNA. Hybridization to sense DNA is significant (maybe
> 10% of hybridization to anti-sense DNA).  We've washed these blots at
> extraordinarily high stringency (like 0.01x SSC, 0.1% SDS, 68 C) and still have
> hybridization to sense DNA (BTW, this is all being done using M13 ssDNA on
> filters).
> 
> Any comments on how we might reduce this noise?
> 
> Rick Alston, Associate Director		rla at canctr.mc.duke.edu
> Molecular Biology Database Facility	Voice: 919.684.5232
> Duke Comprehensive Cancer Center	FAX: 919.684.5296
> DUMC 3424, Durham NC 27710
> 
> 

Hello Rick:

I've had similar trouble with hot RNA sticking nonspecifically to filter-
immobilised DNA; like you, I found super-stringent washing was not much help.
The following procedure did solve the problem though. The key step is a
digestion of washed filters after hybridization in RNAse A. Regions of 
probe strands that are truly and thoroughly hybridized to DNA are not digested
since they ae double-stranded. Hot strands that are just nonspecifically stuck
become digested and removed.

The procedure I used is a modification of that outlined by Lynn et al. (1983)
PNAS 80:2656-2660.

Hybridize as with DNA probes: 5X Denhardts, 5X SSC, 0.1% SDS, 50% formamide,
except:
1. pre-hyb 1 hr. in hyb soln + 100 ug/ml unlabeled tRNA at 55 degrees;
2. hyb with labeled RNA at 55 degrees;
3. wash 3X (5 min each) in 2XSSC, 0.1% SDS, room temp;
4. wash 2x (5 min each) in 2xSSC only, room temp;
5. incubate 15 min in 2XSSC + 1 ug/ml DNAse-inactivated (i.e., boiled/renatured)
	RNAse A;
6. wash in 0.1XSSC, 0.1% SDS, 30 min, 50 degrees. Dry & film as usual.

I hope this helps. 
Cheers,
Kevin.

-- 
==========================================
Kevin W. Ryan
Department of Virology & Molecular Biology
St. Jude Children's Research Hospital
Memphis, Tennessee, U.S.A.
  phone (901) 522-0411, fax (901) 523-2622
    Internet: ryan at mbcf.stjude.org
==========================================