the tubes were shaken vigorously by hand for 15 seconds and then incubated at room

temperature for 2-3 min. The samples were then centrifuged at 12,000g for 15 min at 2-

80C. This separated the solution into an aqueous phase containing the RNA and an

organic phase containing DNA. The colorless upper aqueous phase was transferred to an

RNase-free tube. The RNA was precipitated by the addition of 0.5 mL propan-2-ol. The

samples were then incubated at room temperature for 10 min, followed by centrifugation

at 12,000g for 10 min at 2-80C. The RNA precipitate was now visible as a gel-like pellet

on the side and bottom of the tube. The supernatant was removed and the RNA pellet was

washed once with 1 mL 75% ethanol. The sample was vortex-mixed and centrifuged at

7,500g for 5 minutes at 2-80C. The RNA pellet was left to air-dry for a few minutes

following decantation of the ethanol. The RNA was dissolved in 100 CIL RNase-free

water for intestine, and 200 CIL RNase-free water for liver (since the solution in this case

appeared to be more concentrated), by passing the solution a few times through a pipette

tip. The solution was then incubated for 10 minutes at 55-600C. The samples were stored

at -800C. The purity of the RNA was checked by running the sample on 1% agarose gel

(with 9.5% formaldehyde) and 10x MOPS buffer. Bands corresponding to the 28S and

18S ribosomal subunits were observed. The purity of the RNA was also checked by

diluting the sample in 10mM Tris HC1, pH 7.5 and measuring the A260/A280 absorbance

ratio (ideally should be between 1.8 and 2.1i).

 DNase treatment of RNA samples. This procedure was done in order to remove

contaminating DNA from RNA preparations, and to subsequently remove the DNase and

divalent cations from the sample. Portions of the RNA solutions were diluted to 100

Clg/mL with RNase-free water. The Ambion 9 (Austin, TX) DNA-removal kit was used.