ERA MODERNA

2.3.1 Modification of the method of Chomczynski and Sacchi

The denaturing solution used was 4M guanidinium thiocyanate, 25mM sodium citrate pH 7, 1% w/v N-laurylsarcosine and 0.72% v/v 2-mercaptoethanol.

RNAzol A was prepared by mixing 1 volume of the denaturing solution with 0.1 volumes o f 2M sodium acetate pH 4 and 1 volume o f water saturated phenol. A 200pl volume of sample was added to 800pl of RNAzol A. lOOpl of chloroform was added, the solution vortexed for 15 seconds, cooled on ice for 15 minutes and the phases separated by centrifugation at 15000xg for 15 minutes at 4°C. The upper

aqueous phase was removed, an equal volume of propan-2-ol added and the nucleic acid precipitated overnight at -20°C, 3pg of glycogen was used as a carrier. The RNA was pelleted at 4°C and 15000xg for 15 minutes. The pellet was washed twice with 1ml o f ice cold 75% ethanol by vortexing and subsequently re-spinning at 4°C and 15000xg for 8 minutes. The pellet was then left in an open tube at room temperature for 15 minutes for any remaining ethanol to evaporate. The dry pellet was resuspended in 20pl o f RNase free water (Chomczynski and Sacchi, 1987).

2.3.1.1 RNA extraction from purified virions, serum, plasma and tissue culture fluid

Sucrose density gradient fractions o f purified virions were mixed with an equal volume of 4M guanidinium thiocyanate, 20mM sodium acetate pH 5.2 and frozen at -70°C. The fractions were received frozen on dry ice, rapidly thawed at 37°C and 400pl of this (equivalent to 200pl of sucrose fraction) extracted. The buffer was adjusted and the RNA extracted according to the modification of the method of Chomczynski and Sacchi detailed above.

For serum, plasma and tissue culture fluid a 200pl volume was extracted according to the method detailed above.

2.3.2 Modified SNAP™ RNA extraction

Alternatively, RNA was extracted using the Simple Nucleic Acid Preparation (SNAP™) RNA extraction kit (Invitrogen^^). This extracts RNA by guanidine-HCl / detergent lysis and adsorption to a silica matrix. The composition o f the solutions used are described in Appendix 1. All centrifugations were performed at room temperature unless otherwise stated.

2.3.2 1 SNAP™ extraction of RNA from PBMCs

450pl of binding buffer was added directly to the cell pellet derived from 5ml of blood. If it was a stored frozen pellet the binding buffer was added immediately it was removed from the -70°C and the cells quickly dissolved by flicking the tube. The solution was thoroughly mixed by inversion. ISOpl of TE pH 8 and 300pl of propan-2-ol was added and mixed by inversion 10 times.

Binding, washing and elution

500pl of the above mixture was transferred to the SNAP™ column, centrifuged at 15000xg for 1 minute and the flow through discarded. The remainder was added to the SNAP column and centrifuged at 15000xg for 1 minute and the flow through discarded. The column was washed with 600pl o f Super wash and the flow through discarded. The column was washed with 600pl o f Ix RNA wash and the flow

through discarded. This wash was repeated with a 2 minutes 15000xg spin and the flow through discarded. The bound nucleic acid was eluted by incubating with 135pl o f RNase-ffee water for 5 minutes and centrifuged at ISOOOxg for 1 minute.

DNasing

15pl of lOx DNase buffer and 3 pi (30 units) of DNase I, (RNase free, Boehringer Mannheim Cat. No. 776 785) was added and incubated for 30 minutes at 37°C. 450pl o f Binding Buffer was added and mixed by inversion 6 times. 300pl of propan-2-ol was added and mixed by inversion 10 times.

The binding, washing and elution steps were again performed, but omitting the Super wash, and the DNasing repeated but using 20 units o f enzyme. Finally the binding, washing and elution steps were repeated but the nucleic acid was eluted in 105pl of RNase-free water.

2.3.2.2 SNAP™ extraction of RNA from brain tissue

The protocol was slightly modified for extracting RNA from brain tissue by inclusion o f a chloroform step to aid the removal o f lipids. Once the tissue had been dissolved in the binding buffer and 150pl o f TE pH 8 had been added, an equal volume o f chloroform (SOOpl) was added and the solution vortexed for 15 seconds. The phases were separated by centrifugation at 15000xg for 15 minutes and the

upper aqueous phase removed. 300p,l of propan-2-ol was then added prior to binding the RNA to the SNAP column as detailed above.

2.3.2.3 SNAP™ extraction of virion associated RNA from serum

200-500pl of serum was filtered using 0.45}im spin filters (Nanosep MF™, Flowgen Catalogue No. U3-0126 Ref. ODM45) to remove cells and cellular debris. The serum was centrifuged for 5 minutes at 13000xg (or for further 10 minutes if not all filtered). 150pl of filtered serum was incubated with 10 units RNase ONE^*^ (Promega Catalogue No. M4261) for 30 minutes at 37°C. The filtered, RNased serum was then extracted using the SNAP™ RNA extraction kit.

lOpg o f poly-A RNA (Boehringer Mannheim Cat. No. 108626) was added to the 450pl of binding buffer to act as a carrier. This was added to the serum and mixed by inversion 6 times. 300pl of propan-2-ol was added and mixed by inversion 10 times. The binding, washing and elution steps were performed as above with a single round o f DNasing (30 units). The RNA was eluted with 105pl o f RNase-free water.