ROLES BÁSICO DE CADA PARTICIPANTE

All glassware was baked overnight and electrophoresis tank was treated with RNase away solution to inactivate the RNases. The glyoxal method was used as follows: RNA samples (RNA ladder, HepG2 cells mRNA, and HCV-HCC mRNA) for the blot were mixed with the following reagents in separate tubes

2.7 pi 6M glyoxal

8 . 0 pi DMSO

1 . 6 pi 0.1MNaH2PO4 pH 7.0

3.7 pi RNA (up to 20pg)

Samples were incubated at 50°C for 60 minutes. 3.2.1.2. Agarose gel electrophoresis:

Agarose gel:

The samples were size separated on 1% agarose gels. The gel was prepared by mixing 1 gm agarose with a 100ml solution made with 10 ml Na H2PO4 and 90 ml DEPC treated water. The agarose was melted at high temperature and cooled down before it was poured into the tray.

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Buffer:

The buffer used for electrophoresis was O.OIM Na H2PO4 pH 7.0. It was prepared by adding 900 ml DEPC treated water to 100 ml O.IM Na H2PO4.

Electrophoresis:

RNA samples were cooled down to 20°C and loaded into the gel after adding 4 pi sterile glyoxal gel loading dye (refer to appendix II). The gel was run at 100 V, about 3-4 V/cm. At the end of the run the gel was stained with Ethidium bromide, 0.5 pg/ml. Capillary blotting was carried out using 20x SSC overnight onto a nylon membrane, Hybond N (Amersham). The membrane was marked for orientation and then was baked at 80°C for 2 hours. After baking, the membrane was washed with 20mM Tris-Cl (pH 8.0) at 65°C to remove glyoxal from the RNA.

3.2.1.3. Hybridisation with RNA probe: Probe synthesis:

To synthesise RNA probes, the sequences of T3 and T7 promoters were introduced to the selected sequences via PCR reaction using primers with added 23 nucleotide promoters; T7-40 fGTA ATA CGA CTC ACT ATA GGG TXT TCC CAG TCA CGA C) and T3RP fGAA ATT AAC CCT CAC TAA AGO GAG CGG ATA ACA ATT TCA CAC). Labeled RNA probes were synthesised using Maxiscript, Ambion. This method uses relatively low nucleotide concentrations (0.5 mM each). Higher nucleotide

concentrations are not necessary since, in these reactions, the low concentration of radiolabeled or modified nucleotide present effectively limits the total yield of the reaction. The total concentration of the limiting nucleotide (labeled/modified) should be at least 3 pM for efficient synthesis of full length RNA transcripts of less than 400 nt (more will be needed to synthesize longer transcripts). To make very high specific

Chapter 3_________________________________________________ Confirmation o f differential expression

activity or extensively modified transcripts one should limit or omit any unlabeled limiting nucleotide present. Starting with PCR template of bands 271 and 330 the following reaction was followed:

8.5pl water

2.0pl DNA (PCR template)

2.0pl lOx Transcription buffer

l.Opl lOmM rATP (ribonucleotide) l.Opl 2mM modified rCTP (provided)

l.Opl lOmM rGTP

2.5pl rUTP

2.0pl T7 or T3 RNA polymerase

The transcription reaction was incubated at 37°C for 1 hour.

DNA was inactivated by adding 1 pi DNase I. The contents of the tube were mixed and incubated at 37°C for 15 minutes.

Each probe was diluted up to lOOpl with DEPC treated water. Purification was done with Sephadex G50 columns as before. Prehybridisation:

1 0 ml solution was prepared for a small northern hybridisation as follows:

3.6 ml DEPC treated H20

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5.0ml 2Ox SSPE

0.8ml 5Ox Denhardt’s reagent

0.2ml 10% SDS

0.4ml yeast RNA 5000 |ig/ml The membrane was prehybridised at 65°C overnight. Hybridisation:

One pi yeast RNA was mixed with the probe then added to the membrane after aspirating an equal volume of the prehybridisation fluid. The membrane was placed in an airtight sealed bag as before and left in a shaking water bath at 65°C overnight. Washing:

The blot was washed twice in 500ml 2x SSPE for half an hour at 42°C in a shaking water bath. The membrane was wrapped in Saran wrap, exposed to an X-ray film and stored at -70°C as before.

Probe removal:

Strip-EZ RNA strippable removal kit, Ambion, was used to remove the probe as follows:

45ml 0.1% SDS in DEPC treated water was used for 3 washes, 15 ml each.

First wash : 200x probe degradation buffer was diluted to Ix with 0.1% SDS making 15 ml solution. The blot was incubated in wash 1 solution for 10 minutes at 6 8°C then the solution was discarded.

Second wash: lOOx Blot reconstitution buffer was diluted to Ix with 0.1% SDS making 15 ml. the blot was incubated for 10 minutes at 6 8°C then the solution was discarded.

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Third wash: This wash was carried out using the remaining 15 ml 0.1% SDS for another 10 minutes at 6 8°C. Then the blot was stored for future use.

3.2.1.4. Hybridisation with DNA Probe:

Seven candidate sequences were selected for probe synthesis, 333a, 353, 361, 367b, 373, 383b and 403. The selection was based on their homology with known genes in the nucleotide sequence database (refer to chapter 2 table 2.3).

Probe synthesis:

The DNA template was a PCR product inserted into pCR2.1 plasmid and digested with EcoRI. The digested DNA was purified on low melting point agarose electrophoresis. The purified DNA was denatured by boiling for 5 minutes then quickly placed onto ice. The DNA probe was transcribed as follows:

5.0pl Oligonucleotide labelling buffer (OLE) l.Opl ESA (lOmg/ml)

16.0pl Eoiled DNA 2.5 n ” P dCTP

0.5pl Klenow polymerase

The tubes were left at room temperature overnight.

Each probe was diluted with 75pi TNE buffer (appendix II).

Each probe was purified through a Sephadex G50 columns equilibrated with TNE buffer, then 12.5 pi boiled single stranded DNA (salmon sperm DNA) were added and the tube was put onto ice.

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Prehybridisation:

The blot was prehybridised in 15 ml prehybridisation solution prepared as follows:

0 . 6 ml water

4.5ml 20x SSC

1.5ml 5 Ox Denhardt’s reagent 0.75ml 10% SDS

7.5ml Formamide

0.15ml ss DNA lOmg/ml (boiled for 5 minutes)

The membrane and the fluid were put in an airtight sealed plastic bag and incubated in a shaking water bath at 42°C overnight.

Hybridisation reaction:

2ml of the prehybridisation fluid were replaced with the radioactive probes. The plastic bag was again sealed airtight and incubated in a shaking water bath at 42°C overnight. The hybridisation fluid was aspirated then the membrane was washed twice in 2x SSC at 65°C for 30 minutes, then once with O.lx SDS for another 30 minutes.

The membrane was wrapped in Saran wrap, labelled with RAD tapes for orientation and exposed to X-ray film at - K fC overnight.

3.2.2. RPA: