RNASE PROTECTION

[MCDONALD at PSL.WISC.EDU]

I'M HAVING A PARTICULAR PROBLEM WITH SOME RNASE PROTECTION EXPERIMENTS THAT 
I'VE BEEN CONDUCTING.

NEARLY EVERYTHING SEEMS TO BE GOING ALRIGHT EXCEPT THAT THE ARG SHOWS A RATHER 
SIZABLE POPULATION (CA. 30) OF PROTECTED FRAGMENTS -- THE LARGEST OF WHICH 
IS THE SIZE OF THE EXPECTED PROTECTED FRAGMENT.  WHILE THE EXPECTED FRAGMENT 
IS THE MOST ABUNDANT, THE SMALLER ONES ARE FAR FROM SUBTLE.  ALL BANDS 
WITHIN A LANE SHOW THE EXPECTED DOSE RESPONSE WITH RESPECT TO TARGET RNA 
CONCENTRATION (WHICH IS THE MAJOR REASON I UNDERTOOK THIS PROTOCOL) BUT I 
FEEL I NEED TO SURMOUNT THIS PROBLEM OF NUMEROUS BANDS BEFORE I HAVE 
PUBLISHABLE RESULTS.  DOES THIS PROBLEM SOUND FAMILIAR TO ANYONE OUT THERE?

I SHOULD ADD THAT PURIFYING THE RIBOPROBE (BY PREPARATIVE GEL ISOLATION) DOES 
NOT HELP THIS PROBLEM.  I'M USING BOTH RNASE A AND T1 IN THE RNASE DIGESTION 
STEP AND HAVE TRIED A VARIETY OF DIFFERENT INCUBATION TIMES AND TEMPERATURES 
-- SAME RESULT.  THE PARTICULAR PROCEDURE I'M USING IS THE ONE FROM CURRENT 
PROTOCOLS AND I HAVE ADHERED TO IT FAIRLY RIGOROUSLY TO DATE.

HELPFUL ADVICE WILL BE GREATLY 
APPRECIATED.