Isolation of Total RNA from just about anything

[Anthony C.]

In article <2ulsl3$esu at agate.berkeley.edu>,
Winoto lab <lab_winoto at maillink.berkeley.edu.> wrote:
>RNase-All total RNA Isolation						Astar Winoto
>										Paul W. Diaz
>										Elicia Penuel
[Deleted RNA extraction protocol]
I am using essentially the same protocol (current protocols in molecular
biology) and its good to see that I can probably leave out one of the
precipitation steps with good results.  I can typically get about 30
micrograms of total RNA from 5 million cells and an A260/A280 of about
1.8-2.0.  My problem is that although I am reading RNA in the spec I
simply cannot get the RNA to run on a gel.  I am using the following 
recipe :

11 ul of RNA (10 ug!!)
9 ul of formaldehyde
5 ul 10X MOPS
25 ul formamide
5 ul of premade gel loading buffer from SIGMA

The gel is

.5 grams of wide range agarose from SIGMA
36 ml of depc treated double distilled water
5 ml of 10X MOPS
9 ml of formaldehyde
5 ul of 500 ug/ml EtBr

The gels are a little too soft (pouring buffer on sometimes rips the
wells) so Im going to halve the formaldehyde today.  The 5 ul of EtBr
is a little too bright so Im going to use 2 ul.  The MOPS is a 10X powder
from sigma that you just add 1 liter of water to (minimize the things that
can go wrong :) ).

The problem is that I cant get my isolated RNA or an RNA marker
(RNA marker III from boehringer mannheim, .3-1.6 bp approximately) to
show up on the gel.  At this point I dont care if the prep is partially
digested with RNAses.. I just want the smear!  If you have any suggestions
let me know!  Thanks a lot. (Someone suggested I use a first strand 
cDNA synthesis kit to make cDNA, then run that out hehe..)  Oh.. the reason
Im isolating RNA is I eventually hope to purify the mRNA and make a 
cDNA library.  Im quite amazed that I cant get the gel to work.  One last
thing is that I want to know how critical the percent of formamide and
formaldehyde in the sample solution is.  Sometimes I use a larger
volume of RNA (say 30ul) how which dilutes the formamide and formaldehyde.
I know they are supposed to remove the RNA secondary structure, but at
this point Im not planning to do any hybridization so the secondary structure
isnt too much of a concern.  Thanks in advance!