CONOCIMIENTOS PREVIOS DE LOS ALUMNOS

RNA extraction

Cells were cultured and harvested as described in the previous chapter.

Total cellular RNA was extracted from fresh or DMSO frozen cell pellets using the

method published by Wilkinson (1988) and described below.

Cells (10^ to 10^ cells) were pelleted and resuspended in ice cold tris-saline (400pl).

After centrifugation at 3000 rpm (4°C) for 30 seconds the supernatant was discarded.

The remaining pellet was resuspended in 400pl ice cold tris-saline and lOOpl ice cold

NDD buffer, each tube was gently inverted ten times and then centrifuged at 3000 rpm

(4°C) for 30 seconds. The supernatant was transferred to a tube containing 500pl 1:1 phenol:chloroform, with 25pl 20% SDS and 15pl 5M NaCl. This was vortexed briefly

and centrifuged at 13 000 rpm (room temperature) for two minutes. The upper aqueous

layer was subjected to further extractions with an equal volume of phenol:chloroform

(without SDS or NaCl) until the interface between the phases was clear. A final

chloroform extraction removed traces o f remaining phenol. RNA was precipitated from

the aqueous phase by the addition of 1/10th volume 3M Sodium acetate (pH 5.5) then

2.5 volumes of ice cold ethanol. This was left at ‘20°C for a minimum of 2 hours (or up

to overnight). Samples were spun at 13 000 rpm for 15 minutes, the pellet washed with

80% ethanol in DEPC water and then air dried until all traces of ethanol had just

evaporated. (Overdrying makes the RNA hard to redissolve fully). RNA was then quantified spectroscopically at CD 260nm, where an CD of 1 is obtained from a RNA

concentration of 40pg/ml . This was done at 1/100 dilution (i.e. lOpl RNA in lOOOpl

Tris-saline 25mM Tris. HCl pH 7 .4 ,130mM NaCl, 5 mM KCl.

NDD buffer 1% Nonidet P-40, 0.5% Na deoxycholate, 0.01% dextran sulphate (made

up in Tris-saline)

DEPC w ater 0.1% Diethyl pyrocarbonate in distilled water, left to stand overnight

then autoclaved.

Gel electrophoresis and RNA transfer

RNA was size-separated by electrophoresis through a denaturing agarose gel containing

formaldehyde. lOpG total RNA per well was loaded and the gel was run at 30mV overnight (18 hours). A photograph o f the gel (with ruler) taken under UV illumination

showed positions of the IBS and 28S ribosomal RNA and the wells. The gel was then soaked in two changes of 10 x SSC for 20 minutes. The RNA was transferred from the

gel to a nylon membrane (Zeta-probe, Promega) by capillary transfer (conventional

northern blotting) using 10 x SSC at room temperature overnight. The gel was viewed

under UV illumination to confirm complete (or near complete) transfer o f RNA. The

RNA was covalently bound to the nylon membrane by UV crosslinking and stored at

4°C.

1% Gel: Ig agarose, lOmls 10 x MOPS buffer, 87mls DEPC water, melted and

allowed to cool to 60°C, S.lmls 37% formaldehyde added in fume hood,

gel poured and allowed to set in fume hood.

10 X M OPS 0.2M MOPS (3-(N-morpholino)-proanesulphonic acid), 50mM sodium

acetate, lOmM EDTA adjusted to pH 7.0 and autoclaved.

Sample buffer: lOpl formamide, 2.5pl formaldehyde, 2.4pl 5 x MOPS buffer

* lOpg RNA in lOpl DEPC water, plus 15pl sample buffer. Denatured at 65°C for 15 minutes and then loaded with 2pl running buffer and Ip l ethidium

bromide ( 1 mg/ml).

Running buffer:50% glycerol, ImM EDTA (pH 8.0), 0.25% bromophenol blue, 0.25% xylene cyanol FF.

Probe preparation and labelling

MDR 1 probe

Supplied by Prof. Borst (The Netherlands Cancer Institute), as the 5' 1.33 Kb of human

MDR 1 cDNA clone 1.7 described in van der Bliek et al (1988) from position '98 to the

EcoRl site at position 1237 with respect to the ATG at position 425 o f Chen et al

(1986). This had been cloned into the EcoRl site of the Pro mega vector pGEM3Zf (in

the sense orientation with respect to the T7 promoter). The insert is specific for the

human MDR 1 gene at high stringency (65°C, 0.1 x SSC)

Preparation o f plasmid DNA

MDR 1 plasmid containing E Coli were grown on L-agar plates containing 0.01 mg/ml

by centrifugation at 3000rpm for 5 minutes, resuspended in lOOpl lysis buffer and

incubated on ice for 10 minutes. Then 200pl of freshly made detergent solution (0.2M

NaOH, 1% SDS) was added, followed by a further 10 minute incubation on ice. After

this 150pL 3M sodium acetate (pH 4.8-5.5) was added, the tube centrifuged (3000rpm,

10 minutes), and the supernatant collected. DNA was extracted by phenol/chloroform

extraction and collected by ethanol precipitation as previously described.

Restriction enzyme digestion

Restriction enzyme digestion was carried out according to the manufacturers'

instructions using the supplied buffers. EcoRl digestion released the 1.33Kb insert

encoding part of the MDR 1 cDNA. BamHl digestion linearised the vector such that transcription from the T7 promoter gave a 1295bp riboprobe in the sense direction (i.e.

sequence identical to the MDR 1 mRNA). Bglll digestion linearised the vector at the opposite end of the insert such that transcription from the SP6 site gave a 984bp

riboprobe in the antisense direction (i.e. sequence complementary to the MDR 1

mRNA).

For preparation of the template for riboprobe synthesis, 20pg of plasmid DNA was

digested with the appropriate restriction enzyme then the DNA was purified using the

Magic Prep system (Promega). The latter works on the principle of selective binding of

the DNA to a particulate resin followed by multiple washes and then elution of the DNA

at low salt concentrations into a small volume of water. Linearised template was stored

Lysis buffer 50mM glycine, 25mM Tris.HCl pH8, lOmM EDTA, 2mg lysozyme.

L -agar Bacto tryptone lOg, sodium chloride 5g, yeast extract 5g, agar 15g to

make 1 litre, sterilised by autoclaving.

Terrific brothBacto tryptone 12g, yeast extract 24g, glycerol 4ml, KH2PO4 2.3 Ig,

K2HPO4 12.54g to make 1 litre, sterilised by autoclaving.

Ampicillin Was dissolved in sterile water, filter sterilised and added to agar at lOpg/ml.

Actin probe

This is a 700 bp Hindlll / EcoRl fragment of the p actin cDNA cloned into pBluescript.

The insert was released by double digestion with EcoRl / Hindlll, gel purified and 50 -

lOOng aliquots were used as a template for probe synthesis.

Probe labelling

The probes were labelled by random priming using the Stratagene PRIME-IT II kit.

This enables synthesis of oligonucleotide sequences incorporating radiolabelled

nucleotide by the Klenow fragment of DNA polymerase I. Labelling was carried out

according to manufacturers' instructions. ^^P CTP with dCTP buffer (containing no

'cold' CTP) was used. The percentage incorporation of radionucleotide into DNA was

bases. Incorporation of greater than 30% was accepted as giving workable probe.

Unincorporated nucleotides and protein were removed by passing the reaction mixture

through a Sephadex column ('Nick column', Pharmacia) with TNE (lOmM Tris pH8,

ImM EDTA, lOOmM NaCl).

Hybridisation

The nylon membrane was prehybridised at 65°C for 10 minutes in Church and Gilbert

hybridisation mixture with denatured sonicated salmon sperm at 200pg/ml. The probe

was denatured (5 minutes at 95°C) and added to the hybridisation solution.

Hybridisation took place overnight (18 hours) at 65°C in rotating glass bottles.

After hybridisation the filters were washed to remove non-specifically bound probe. The

initial wash was in 2 x SSC, 0.1% SDS at 65°C for 10 minutes, followed by 2 washes in

0.5 X SSC, 0.1% SDS at the same temperature. This was followed if necessary by a

higher stringency wash in 0.1 x SSC, 0.1% SDS.

Excess wash solution was blotted off the filters which were then wrapped in Saran wrap

for autoradiography.

Church and Gilbert buffer 0.5M Na2HP0 4 (pH 7.2, with orthophosphoric acid), 7% SDS, 0. ImM EDTA

Autoradiogaphv

Autoradiography of 32P-labelled probe bound to membranes was carried out using Fuji

RX medical X ray film in cassettes which had tungsten intensifying screens. Plastic

wrapped filters were placed in direct contact with the film and exposed at '70°C for