RNA extraction reagents a. Preparation of solutions 1M Tris Dissolve 121.

1g of Tris base in 800ml of H2O Adjust the pH to the desired value by adding concentrated HCl pH HCl 7.4 ~70ml 7.6 ~60ml 8.0 ~42ml 0.5M EDTA pH 8.0 Add 186.1g of disodium EDTA to 800ml H2O Adjust the pH to 8.0 with NaOH (~ 20g NaOH pellets) L6 Buffer guanidinium isothiocyanate (GTC) 0.1M Tris-HCl pH 6.4 0.2M EDTA pH 8.0 Triton X-100 L2 Buffer guanidinium isothiocyanate (GTC) 0.1M Tris-HCl pH 6.4 70% ethanol Ethanol distilled H2O b. 1. 2. 3. Preparation of size fractionated silica Add 60g silicon dioxide, SiO2 (Sigma; S-5631) to demineralised water to a total volume of 500ml in a measuring glass cylinder. Allow the silica to sediment under gravity for 24h at room temperature Extract 430ml of supernatant and add demineralised water to 500ml and shake vigorously. 70ml 30ml 180g 150ml 60g 50ml 11ml 1.3g

4. 5.

Sediment for 24h at room temperature. Extract 440ml supernatant and add 600ul HCl (32%, w/v) to adjust the silica suspension to pH 2.0. Note: Wear visor, apron and gloves when handling concentrated acids. Aliquot the silica suspension in 4ml volumes in glass bijoux bottles and sterilise by autoclaving.

6. 1.

Add 1 g silicon dioxide, SiO2 (Sigma; S-5631) to demineralised water to a total volume of 5 ml in a measuring glass cylinder. 3. Extract + supernatant and add 44,5ul HCl (32%, w/v) to adjust the silica suspension to pH 2.0. Note: Wear visor, apron and gloves when handling concentrated acids. Aliquot the silica suspension in 4ml volumes in glass bijoux bottles and sterilise by autoclaving. c. 1. Preparation of RNase-free deionised water Add 100ul of diethylpyrocarbonate (DEPC)(Sigma: D-5758) to 100ml of cell culture grade deionised water.

NOTE: Perform the addition in a fume cupboard. 2. 3. Incubate for >12hr at 37oC. Autoclave for 15min.

Prosedur ekstraksi metode boom dari darah 1. L6 900 uL tambah darah 100 uL shaker selama 1 malam 2. ditambahkan suspensi diatom 30 uL setiap tube 3. selanjutnya disentrifuge 12.000 rpm selama 30 dtk supernatan dibuang 4. L2 1000 uL tambah ke tabung + suspensi diatome divortex 10 kemudian disentrifuge 12.000 rpm selama 15 dtk supernatan dibuang dilakukan 2 kali

5. pellet dari L2 ditambahkan 1000 uL etanol 70% divortex 10 kemudian disentrifuge 12.000 rpm selama 15 dtk supernatan dibuang dilakukan 2 kali 6. pellet dari etanol 70% ditambahkan aseton absolute 1000 uL divortex 10 kemudian disentrifuge 12.000 rpm selama 30 dtk supernatan dibuang 7. selanjunya pellet dari aseton di keringkan pada oven suhu 56 C selama kurang lebih 1 jam 8. selanjunya tambahkan RNAse free water 70 uL kemudian vortex 10 dengan pelan sampai homogen selanjunya diinkubasi suhu 56 selama 10 menit selanjunya di sentrifuge kecepatan 12.000 selama 15 9. kemudian supernatan diambil sebanyak 30-40 uL kemudian dimasukkan ke tabung baru. 2.1 Guanidinium isothiocyanate (GTC)/ silica gel extraction of nucleic acid) From Fecal The 'Boom' method [Boom et al., 1990 J Clin Microbiol]. 1. Add 200ul of faecal suspension to 1ml of lysis buffer-L6 (see section 8) buffer and 20ul of size fractionated silica (see section 8) in a 1.5ml screw-capped microcentrifuge tube. RNase-free distilled water is included in each run to act as a negative control. Cellculture grown SA11 or RRV should be used as a positive control1. 2. 3. Vortex for 10 sec and incubate at RT for 15 min. Pellet by centrifugation (microcentrifuge) for 15 sec (13,000rpm), discard the supernatant. (Collect the supernatants for disposal of toxic waste, see Disposal of toxic waste; see section 9). 4. Wash the pellet with 1ml of lysis buffer L2 (see section 8), mix well by vortexing and pellet by centrifugation as before. Repeat with
1

An animal rotavirus is used as a positive control in order to monitor laboratory contamination.

L2 buffer then similarly wash twice with 1ml 70% ethanol and 1ml acetone once2 (store the wash fluids for disposal). 5. After removal of the acetone (perform carefully as pellets may become dislodged) centrifuge and place tube with lid open at 56oC in a dry heating block for 5 minutes. 6. Add 49ul of RNase-free distilled water and 1ul of RNasin, vortex and incubate at 56oC for 15 min to elute the nucleic acid from the silica. 7. Pellet by centrifugation at 13,000rpm for 4 min and extract the supernatant (avoid disturbing the silica. Recentrifuge if silica becomes resuspended). This can be stored in a new microfuge tube at 4oC for 24h or -70oC for longer.

It is important to resuspend the silica completely between wash steps.