Formaldehyde, hybridization, and Northern blots

David S. Huen smh1008 at cus.cam.ac.uk
Mon Jul 18 13:01:46 EST 1994


In article <Ct37I6.G4y at acsu.buffalo.edu>, camhuber at ubvms.cc.buffalo.edu
(Joel Huberman) writes:
>Dear Netters,
>
>	To size-fractionate RNA prior to Northern blotting, formaldehyde gels
>are frequently used.  Formaldehyde reacts covalently with the bases in single-
>stranded nucleic acids, preventing the bases from Watson-Crick base pairing.
>By preventing base pairing, formaldehyde prevents formation of secondary struc-
>ture, so RNAs run according to their size in the gel.  It seems to me, however,
>that the formaldehyde-modified RNA should not be able to hybridize with a probe
>after it has been Northern-blotted to a membrane.  Obviously, it does hybri-
>dize;  otherwise formaldehyde gels would not be used for Northern blottings.
>Thus, the formaldehyde reaction must be reversible.  I would like to know whe-
>ther the formaldehyde reaction is fully reversed under standard hybridization
>conditions, or is there a possibility that use of a formaldehyde gel leads to
>less efficient hybridization than would use of a standard gel or a different
>type of denaturing gel, such as a formamide gel?
>
At one stage, I was using vacuum-transfer onto charged membrane with the
alkali-transfer method and could never get probe to hybridise. On
considering the theory behind the method, I came to the conclusion, which
you have also realised, that the Schiff base adduct must be removed
somehow. At least with the  glyoxal method, the adduct is very unstable at
high pH and so should be removed efficiently in alkali transfer. This is
not the case with formaldehyde-I suspect that this adduct may not be fully
reversible. I managed to get probe to work eventually by boiling the
filters in Tris for 15 min, which presumably offers a alternative amino
group for the formaldehyde formed on removal to react with (speculative,
presumably ammonia solution should work too!). Also, for some strange
reason, use of charged membrane gave high background with formaldehyde gels
unless I went thru the traditional acid/neutralisation step before transfer
(does formaldehyde react with what are presumably amino groups on the
charged membrane itself? if so, the Tris neutralisation step presumably
removes the formaldehyde from the gel before it causes problems). Enough
speculation....all I know is that the kludge-fix works...

-- 
David S. Huen                        Phone: (0223) 333921
CRC Human Cancer Genetics Group      Fax:   (0223) 333875
Dept. of Pathology                   e-mail: smh1008 at cus.cam.ac.uk
University of Cambridge
CB2 1QP                              
United Kingdom



More information about the Methods mailing list