Nucleic acids RNA.

2.1 RNA preparation.

For the preparation of RNA, gloves, sterile plastic, and fresh DEPC-treated and autoclaved solutions were used, and the samples were kept at 4**C or below at all times. Tissue samples or cell pellets were placed directly into 3ml of 3M LiCl/6M urea on ice. The tissue

samples were homogenised with the ultra-turrax on hill power for 1 min. Samples were then

sonicated for 1-2 min to shear the genomic DNA and then left at 4**C overnight. The solution was centrifuged at 14K rpm at 4"C for 30 min. The supernatant was removed and the pellet pipetted back and forth in 500/d of 3M LiCl/6M urea for 1-2 min. The sample was again centrifriged at

4*C for 30min, with the supernatant being removed. The pellet was then resuspended in 300/d of lOmM Tris/0.5% SDS. Foffowing this, 2x phenol/chloroform, plus Ix chloroform extractions were performed. The RNA was precipitated with 30/xl of NaOAc and 800/tl of ethanol. The sample was left on dry ice for 30 min, before it was pelleted at 4®C, 14K rpm, for 30 min. The pellet was washed with 70% ethanol and resuspended in 20-100/il of DEPC-treated water. The samples were stored at -70®C until required.

2.2 Slot blot analysis of RNA.

The slot blot manifold was prepared by soaking it in O.IM NaOH for 30 min, then washing well with DEPC-treated water. The nitrocellulose filter (0.45-micron pore size) was placed in DEPC-treated HgO for 2min, then soaked in 20x SSC for Ihr at room temperature. As for DNA slot blotting, the 2 sheets of 20x SSC-soaked Watman 3MM paper were placed on the lower half of the manifold with the filter on top. The upper half of the manifold was then fitted and clamped in place. The suction line was attached and the vacuum turned on. All the slots were then washed with lOx SSC under suction. Between 5-20/ig of RNA was made up to lO/il in DEPC-treated HgO. To this, 20/d of 100% formamide, 7/d of 37% formaldehyde and 2/d of 20x SSC were then added. The samples were mixed well, and incubated at 6 8®C for 15min. The

samples were removed to ice, mixed with 2 volumes of 20x SSC, and applied to the appropriate slot. After the last sample was loaded, the vacuum line was removed and the filter was allowed to air-dry for 5min. The filter was then baked in an 80*C oven for 2hr, before being hybridised

in conditions as used for a northern blot.

2.3 Northern blot analysis of RNA.

The gel tank and apparatus was first washed well with 0. IM NaOH and rinsed in DEPC- treated HgO. RNA samples (5-30/ig) were made up to 4.5/il in DEPC-treated HgO. To this, 2fi\ of lOx MOPS, 3.5/a1 of 37% formaldehyde, and lOfil of 100% formamide were added and mixed well. The samples were then incubated at 6 8**C for 15min before being removed to ice. Northern

loading dye (0 . 2 vol.) was added, and the samples were loaded onto an agarose (0.8%-

1.2%)/M0PS (lx)/formaldehyde (6%) gel. The samples were run through the gel at 1-5 V/cm

until the furthest blue dye front had reached the end of the gel. Boehringer RNA size standards were used. The gel was then soaked in 50mM NaOH/lOOmM NaCl for 20min, lOOmM Tris pH 7.6 for 20 min, and 20x SSC for 20 min. The gel was then blotted to 20x SSC soaked nitrocellulose of Hybond-N (Amersham) filters in the manner described for Southern blot transfer. After 12-16hr the filter was removed and baked in an 80"C oven for 2hr, and hybridised as described below.

2.4 Hybridisation of RNA filters.

RNA filters were pre-wet in 2x SSC then placed in Hybaid hybridisation bottles. The filters were pre-hybridised for 2hr at 42"C in 20ml of RNA hybridisation solution without the radio-labelled probe. The pre-hybridisation solution was removed. The probe was then added to 20ml of fresh RNA hybridisation solution and incubated overnight at 42®C. The filters were then washed twice for 20 min in 2x SSC/0.1 % SDS and twice for 20 min in 0.2x SSC/0.1 % SDS. The filter was then covered in Saran wrap and exposed on film (4hr-lwk) or on the Molecular Dynamics Phosphorimager.

2.5 SI nuclease protection analysis.

SI nuclease protection analysis was used to detect RNA in the tissues of transgenic mice. 5-30/ig of RNA and lOng of end-labelled probe were mixed, made up to 300/il with HgO, and

precipitated using 30fi\ of 2M NaOAc and SOO^tl of 96% ethanol (10 min dry ice). The nucleic acid was pelleted by centrifugation at 14k rpm, air-dried for 5min, and resuspended well in 15/a1

of SI nuclease hybridisation buffer. The samples were then incubated at 90"C for S min to denature the RNA, and then swiftly transferred to a water bath at S0-55**C. The probe and RNA were allowed to anneal in these conditions for at least 16hr. To each sample 200^1 of ice cold SI digestion mix, containing Ix digestion buffer, 10/xg of tRNA carrier, and 100 units of SI nuclease, was added as the tubes were removed from the water bath. They were quickly sealed, vortexed and placed on ice. After each had been removed from the bath, all the samples were placed at 23*’C for 2hr ISmin. The digestion reaction was terminated by placing the samples on ice. The remaining nucleic acid were phenol/chloroform extracted and ethanol precipitated in the presence of 0.1 vol. of 2M NaOAC. After centrifugation the pellet was resuspended in 5fi\ of SI loading buffer. The samples were then denatured for 5 min at 9 0 ^ , placed on ice, and loaded onto a 7% denaturing polyacrylamide gel. The gel was electrophoresed and visualised as described above.

SI probes: Tat - 610bp Styl fragment from pTZ19 (gift from Dr. E. Blair, Wellcome). Protected fragment is 498bp at 5* end of tat gene.

U6 - 326bp Fokl-Eael fragment from pGEM3Z (gift from Dr. M. Antoniou). Protected fragments of 79bp and 87bp at 5’ end of U6 gene.

2.6 Reverse transcription.

Reverse transcription of RNA (and PCR amplification of the resulting cDNA fragments - see above) was used to analyse the presence of transgenic transcripts in various mouse tissues. 1-lO^g of RNA in 5/tl of H^O was heat denatured for 5 min at 65®C then placed on ice. To this, 2/tl of lOx RT buffer, and 10 units of Super RT (HT biotechnology) were added and made up to 20/d with HgO. The mix was incubated at 42®C for 2hr. 30/il of ImM Tris pH 7.5 was then added and the resulting cDNA solution stored at -20XZ until required.