RNA extraction from Gram negative bacteria

[Rosamund Eira Powles]

I have tried extracting RNA from Thiobacillus ferrooxidans using the method 
for Gram negative bacterium in Current Protocols.  This involves 
resuspending your cells in a STET lysis buffer, followed by a room 
temperature phenol/chloroform step, precipitation of total nucleic acids, 
and a further two phenol/chloroform steps, with a precipitation, the sample 
is then loaded onto a CsCl cushion and the RNA spun down in an 
ultracentrifuge.  I have found this method works very well for E. coli, but 
I got no RNA from Thiobacillus, a colleague also had trouble using the 
method to extract RNA from Vibrio cholera.  I have been able to extract RNA 
from Thiobacillus using the Hot Phenol method of preparation, but as I am 
looking for large transcripts of approximately 8 kb in size, I thought the 
CsCl method would ensure getting intact large transcripts, whilst the hot 
phenol method could lead to nicking of the RNA and shearing.  If anyone has 
any suggestions for extracting large RNA transcripts from Thiobacillus 
ferrooxidans I would appreciate it.  The bacterium is a very slow grower, 
and the cultures are very dilute, so it is very difficult to get large 
quantities of cells.