Reverse Transcriptase Polymerase Chain Reaction

Reverse Transcription

First strand cDNA was synthesised from total RNA using Superscript (Gibco). This

enzyme is purified from E. Coli expressing the cloned pol gene of M-MLV RT from

which the RNase H sequence has been deleted. It uses single stranded mRNA or DNA

as a template in the presence of a primer to synthesise a complementary DNA strand.

RNA (Ipg), oligonucleotides (0.5pg) and water (to make tube volume 11 pi at this

stage) were first incubated at 70°C for 10 minutes. After this O.IM DTT (2pi), dNTPs

(ip g o f lOmM), Superscript enzyme (Ipl) and enzyme specific buffer (4pl of 5 x

buffer) were added and mixed. A one hour incubation at 37°C allowed cDNA to be

transcribed, the reaction was then stopped by heating to 70°C for 10 minutes then

placing the tubes on ice. cDNA was stored until use at '20°C.

Polymerase Chain Reaction (PCR)

This reaction was carried out in an automatic programmed heating block (Hybaid

Omnigene). Appropriate controls were always included, i.e. negative controls were no

RNA and no cDNA tubes, positive control was a known MDR 1 positive cDNA which had been synthesised in the same reverse transcription reaction as the test RNA.

PCR primers were synthesised commercially, and were chosen to be specific for the

B jM Sense ACC CCC ACT GAA AAA GAT GA

Antisense ATC TTC AAA CCT CCA TGA TG

Internal AGT CAC ATG GTT CAC ACG GC

MDR 1 Sense CCC ATC ATT GCA ATA GCA GG

Antisense GTT CAA ACT TCT GCT CCT GA

Internal GGT GCT GGG AAG ATC GCT ACT GAA GCA ATA

Orientation refers to the sequence of the corresponding mRNA. Sequences o f the

primers are read from left to right as 5' to 3'.

The MDR 1 cDNA sequence used is that reproduced in figure 1.4a from Chen et al

1986. The primers and internal oligonucleotide used as a probe are marked. The MDR 1 primer pair spans exons 21 and 22, and amplify a 157 bp fragment which is specific for

RNA (amplification of any contaminating DNA in the RNA preparation would give a

product of 2257kb).

The B2 microglobulin gene sequence used is found in Gussow et al 1987. The primers

and internal oligonucleotide used are marked. These primers span two introns, and

generate a 120 bp product (the DNA product would be 2175bp).

Internal oligonucleotides were used to confirm specific sequence amplification as

described later. The MDR 1 internal oligonucleotide is a 30 bp sequence, containing 50% GC bases, with a Tm of 60.9°C. The B2 microglobulin internal oligonucleotide is a

PCR was performed using cDNA (Ipl), 200nM dNTPs,0.5pM primer pairs, Taq

enzyme (0.1 pi) with 2.5pl 10 x buffer (lOOmM Tris.HCl pH 8.3, 500mM KCl, 0.1%

gelatin) and water (to make tube volume up to 25pl). Two drops o f mineral oil were

added to each reaction tube to minimise evaporation during the reaction. Appropriate

positive and negative controls were included in each experiment.

The PCR program used gave a 3 minute initial dénaturation at 94°C, followed by 1

minute at 57°C (to allow DNA to anneal with oligonucleotides), 2 minutes at 72°C (to

extend the new double stranded molecule). This first cycle was followed by a total o f 39

cycles identical to the first except for a shorter initial dénaturation (30 seconds at 94°C).

Gel elecfiopboresis of PCR products

For PCR products of this size a horizontal 3% Nu Seive agarose gel run at 70mV for 3

hours provided adequate resolution of the two products when run together. 1 x TAB was

used as the tank buffer. A 5 pi aliquot of the PCR product was sufficient to determine

the success of a PCR reaction, however for the purposes o f subsequent capillary transfer

and hybridisation a 20pl aliquot was used. A 1 Kb DNA size ladder (Gibco) loaded onto

each gel allowed accurate size estimation of the bands. A photograph o f the gel taken

Avith UV illumination showed positions of the PCR fragments.

Gel: 3% Nu Sieve agarose in 1 x TAB with 2.5pl Bthidium Bromide

(lOmg/ml) made to 5Omis for mini and lOOmls for midi gel.

50 X TAE 242g Tris base, 57.1 ml glacial acetic acid and 100ml 0.5M BDTA (pH

Gels to be hybridised with the internal oligonucleotide probes were run as midi (100ml)

gels in the same way as above.

Southern blotting

The PCR products were transferred from the gel to a Zeta-probe (Promega) nylon

membrane by capillary transfer in 0.4M NaOH. The gel was viewed under UV

illumination to confirm complete transfer of DNA. This did not need to be UV cross

linked. The membrane was stored at 4°C until required for hybridisation.

End labelling of oligonucleotide probes

The internal oligonucleotides described above were used to confirm the presence of the

correct, specific PCR products. These oligos were end labelled with ^^P ATP in a phosphate transfer reaction catalysed by polynucleotide kinase. This was achieved using

lOpmol oligo, 2pl 10 x buffer (One Phor all PLUS) 15pl water, Ipl ^^P ATP and Ipl

PNK enzyme, these were mixed and incubated for one hour at 37°C. Unincorporated

nucleotides and protein were removed by passing the reaction mixture through a 'Nick

column' with TNE as described previously.

Hybridisation

The nylon membrane was prehybridised at 55°C for 10 minutes in Oligo-Hyb buffer.

The 32P-labelled oligonucleotide probe was then added to the hybridisation buffer.

Hybridisation took place overnight (18 hours) at 55°C for the MDR 1 internal and 45°C

for the B2 microglobulin internal probe.

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

depending upon which probe was used), 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 high

stringency wash in 0.1 x SSC, 0.1% SDS. The filters were blotted and wrapped in Saran

wrap for autoradiography.

Oligo-Hyb buffer 0.1% SDS, 0.1% Sodium pyrophosphate, 0.05% BSA, 0.05%

PVP, 0.05% Ficoll, 5 X SSC.

Autoradiography