RNA folklore? - Reply
varyc.mmcri at OFFICE.MMC.ORG
Wed Dec 14 08:04:43 EST 1994
1 various conditions will deactivate ribinucleases with some degree of reversibility
depending on the rnase; al rnases are not panc rnaseA. diethylpyrocarbonate
irreversibly ethoxylates rnase A and irreversibly inactivates this rnase and others.
Your mRNA source (ie fruit) may be blessed with more labile rnases.
I'm also reminded of my post doc days when I did RNA structure work; we all in the
lab took individual tubes of 5' end labeled globin mRNA in TE (10,000 cpm),
placed our ungloved thumb(s) over the end of the tube and inverted the tube once
for 5 sec. (then washed hands). The RNA was mixed with carrier tRNA,
precipitated, and run on a sequencing gel. This "individual rnase assay" showed
that some samples were almost intact (mine) while others were gone. This result
was reproducible. So that's why we spend all this money on latex gloves.
20ug/ml RNA in bicarbonate buffer, pH 9.0, 5min at 80degC gives a single hit
ladder with end labeled RNA, ie <=30% degraded.
10mM NaOH on ice for 5min does about the same. Your 50mM NaOH conditions
are designed to bust up the RNA to small probable fragments. Remember that the
concern used to be to keep the RNA completely intact (RT, structure mapping).
hope these reflections help.
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