RNA preparation from heart???

Harry Witchel Harry.Witchel at bristol.ac.uk
Mon Nov 6 07:16:13 EST 1995


Hello!
	I am trying to prepare RNA from rabbit heart for RT PCR.  Does anyone 
have a method they used in heart tissue which produces clean RNA?  So far I 
have tried two methods, each not quite working correctly:

1)  Single tube method.  1 gm tissue, frozen (liquid N2), ground, and dounce 
homogenized in 10 ml of GIT solution "D" (includes acetate, Tris, EDTA, and 
BME), added 1 ml of 2 M NaOAc ph 4.0, mixed with H2O saturated phenol (10 ml), 
then mixed with 2 ml chloroform, spun, harvested aqueous layer, precipitate 
with equal amount of isopropanol.  So far, so good, a fairly visible white 
pellet (50-100 ug of something, probably DNA and RNA).  Then I resuspended 
pellet in GIT solution again, reprecipitated in  isopropanol, wash with 70% 
EtOH, dry briefly, and resuspend in 100 ul of H2O (DEPC treated).  First of 
all it did not seem to go back into solution for 15 minutes, but then later 
the entire solution became one gelatinous mess which could not be pipetted 
(liquid would go into the pipet tip, but as I removed the tip from the tube, a 
single strand of fluid (DNA?) would extend from the tube to the tip, and 
finally the strand would pull the fluid out of the tip.

2) Cesium trifluoroacetate.  Again 1 gm tissue, freeze, grind, GIT 3 mls.  
Spin out insolubles, and layer homogenate/GIT on top of a step gradient (CsTFA 
bottom (.7 ml) rho =1.75, middle (.6 ml) rho = 1.5, top (3 ml) is GIT).  Spin 
overnight at 40 K rpm swinging bucket 12 degrees C, and sure enough there is 
DNA floating between the two layers and RNA pelleted at the bottom.  I 
carefully pipet out everything except the bottom .3 ml.  I cannot pour off the 
CsTFA because the RNA pellet always dislodges (standard Sorvall 5 ml tubes, 
pretreated with DEPC, washed extensively, and autoclaved).  So I simply 
suspend the RNA dislodged pellet in the CsTFA, transfer the colloid to an 
eppendorf tube, and respin the pellet.  The fluid is removed, the pellet is 
washed with 70% EtOH, and the pellet is dried and resuspended again in GIT 
solution, whereupon it is precipitated again with Isopropanol.  After washing 
and resuspension, the RNA has two problems: it is hard to load on the gel 
because it floats out of the lane (meaning organics are still present, but I 
thought I had dried the pellet after the ethanol, and there  are no other 
organics), and also when I do get it into a gel, there is a bright band in the 
loading well (high salt?) as well as a smear running down the length of only 
the center of the lane until it reaches the RNA region.

I should apologize as well for my gels which are nondenaturing 1% agarose 
gels, but I find that formaldehyde makes detection very difficult (have to 
load 10X as much sample) and formaldehyde gels are very slow, so I just 
pretreated my RNA samples with heat to denature them.
	Harry




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