RNA dot blot - is the denaturation step important? (Mar/27/2006 )

hi everyone,
I am doing RNA dot blot for the first time. Because i could't find formamide in my lab. I just heat the RNA(in H2O) then spot it onto nylon membrane. After crosslinking, and staining with methylene blue, the RNA was right there on the membrane. Does this mean that I can proceed to the next step?

-WHR-

hi
normally, denaturation is done by heating 56° 10' your sample.
if the step is done and if you've succesfully crosslinked the RNA, ou can go to next step

-fred_33-

QUOTE (fred_33 @ Apr 2 2006, 10:13 AM)

hi
normally, denaturation is done by heating 56° 10' your sample.
if the step is done and if you've succesfully crosslinked the RNA, ou can go to next step

Thank you, fred,
I want to investigate the target gene expression level by RNA dot blot using dig system(roche). As a positive control, I spot plasmid containing the target gene cDNA. I can detect signal as few as 0.1ng plasmid. The sensitivity is really not satisfactory, right? Maybe I need to optimize it.

-WHR-

hi
by northern blot, i've detect 2ng of complementary probe. 0.1ng seems not so bad...
if you think it's a pb, try to check labelling of the probe.

-fred_33-

QUOTE (fred_33 @ Apr 3 2006, 07:04 AM)

hi
by northern blot, i've detect 2ng of complementary probe. 0.1ng seems not so bad...
if you think it's a pb, try to check labelling of the probe.

hi fred,
The sensitivity could be a problem. Assume that the total RNA contain 2% mRNA, which in turn contain 0.2% of a high expression mRNA species, for example, beta-actin. You may have to load approximately 50 ug total RNA to detect a beta-actin band. right?

-WHR-

in "classical northern" 15µg total RNa may be enough.
But for dot blots, publications shows a 50µg loading.

-fred_33-