Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

RT-PCR experiments in Chapter 4 were performed by Dr. Dagmar Alber, whilst RT- PCR reactions for probes for Northern blots for Chapter 5 were carried out by myself.

For the purposes of generating probes for Northern blots, sEnd. 1 cells in 6 well

plates were exposed to SOOjal of lOpg/ml o f LPS {Salmonella typhosa - a gift from

Neale Foxwell) diluted in growth media, and were suspended in TRIZOL after 2

(approximately 4pg total RNA) of this solution was used for the RT-PCR reaction, using GibcoBRL reagents. Ipl of Oligo(dT) was added to 2pl o f RNA and made up to 12pl with DEPC. The mixture was heated to 70°C for 10 minutes and chilled on

ice. To this was added 4)l i1o f 5xFirst Strand Buffer (250mM Tris-HCl pH 8.3, 375

mM KCl, 15mM MgC12), 2pl of O.IM DTT and Ipl o f lOmM dNTP mix (lOmM each of dATP,dGTP, dTTP and dCTP). These reagents were mixed gently and kept at 42°C for 2 minutes. Ipl o f SUPERSCRIPT It (Reverse Transcriptase) was then added, and the mixture then incubated at 42°C for 50 minutes. The reaction was inactivated by heating to 70°C for 15 minutes and then placing on ice.

2 pi of the first strand reaction mixture was then used for the subsequent PCR. To this DNA template the following were added: lOpl o f PCR Buffer (200mM Tris-HCl

pH 8.4, 500mM KCl); 3pi of 50mM MgCl2; 2pi dNTP mix; Ipl o f each primer; Ipl

Taq DNA polymerase (5U/pl); and 80pi of distilled water. The mixture was

denatured at 94®C for 3 minutes, and then 38 cycles of 94°C for 30seconds, 55°C for 30 seconds and 70°C for 30 seconds were carried out. Finally the mixture was held at 72°C for 10 minutes and then kept at 4°C.

The PCR product was mixed with Gel Loading Solution (Sigma) in a 6:1 ratio, and loaded onto a 2% agarose gel with ethidium bromide. Bands were visualised under UV and cut out with a sterile scalpel. Gel extraction was carried out using a QIAQuick Gel Extraction Kit (Qaigen).

Dr. Alber used Perkin-Elmer reagents with an anchored oligo(dT). The PCR conditions were 94°C for 4 minutes and then 30 cycles at 94°C for 1 minute, 56®C for 1 minute and 72°C for 1 minute. The final cycle was 72®C for 7 minutes.

2.6.6 Labelling of probe

DNA probes were labelled using a Random Primed DNA labelling kit (Roche

Molecular Pharmaceuticals). 2j l i1o f DNA probe eluted by the QIAQuick Extraction

kit was added to lOpl o f distilled water and denatured by heating to 1I0°C for 10 minutes before being chilled on ice. To this Ip l of dATP, dTTP and dGTP (0.5mM in Tris-buffer) were added plus 2pi o f hexanucleotide mix (in lOx buffer) and 2pl o f Redivue ^^P dCTP (Amersham Pharmacia Biotech), and Ipl o f Klenow enzyme (2U/pl). The mixture was mixed by pipetting and transferred to a water bath at 37°C for 1 hour.

The probe was cleaned by passing it down a Nick Column (Amersham Pharmacia Biotech). The storage buffer was allowed to run through the column followed by an equal volume of TE buffer (pH 8). The column was then set up above the first o f a series of three sterile eppendorfs. The probe was removed from the ice and placed in the centre of the column and washed through with 400pl o f TE buffer. This was repeated twice more with the same volume o f buffer with the Column over the remaining two eppendorfs. Each tube was checked for activity using the Geiger Counter, and the second most active tube selected. This probe was then denatured by heating to 110°C for 10 minutes and chilled immediately on ice.

2.6.7 Hybridisation

The Hybond N+ membrane bearing the RNA was prehybridised in a roller tube with 5-lOml of ExpressHyb (Clontech) at 68^C for 30 minutes. Then the ExpressHyb was poured out, and 3-5ml of fresh ExpressHyb was mixed with the probe in a Falcon

tube and added to the roller tube. The blot was then hybridised for 1 hour at 68°C. The hybridising solution was then drained out and the blot washed twice with Wash

1 and Wash 2 for 15-20 minutes each at 25°C and 50°C respectively. Wash 1

consisted o f 2x SSC, 0.05% SDS and wash 2 o f 0. Ix SSC, 0.1% SDS. The blot was then wrapped in cling film. For the probes against mRNA of adhesion molecules the blots were placed on an Imaging Plate BAS-MP (Fuji Photo Film Co. Ltd) for 36 hours, and for the control S I8 probe only for 2-5 minutes, before quantification. Films of adhesion molecule blots were taken by placing blots on BIOMAX-MS film (Kodak Scientific Imaging Film) and keeping at -80°C for 36-48 hours. Meanwhile blots for the control probe were placed on X-OMATAR film (Kodak Scientific Imaging Film) at -80°C for 2-4 hours.

2.6,8 Sequencing of probes

Probes were sequenced using the pre-mix in the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction kit. 8pi o f premix was added to Ip l o f DNA probe and 3.2 pi of a single primer at Ipmol/pl and made up to 20pl with distilled water. This was repeated using the reverse primer. Reaction mixtures were vortexed and kept on ice. The following cycling reaction was carried out for 25 cycles (Techne or Biometra thermocyclers overlaying with silicone oil as necessary):

denatured at 94°C for 30 seconds, annealed at 55°C for 18 seconds and allowed to

extend at 60®C for 4minutes. Reaction mixtures were then pipetted into eppendorfs containing 2pl o f sodium acetate and 50pl o f 95% ethanol, vortexed and left on ice for 10 minutes. Pellets were precipitated by centrifuging for 30 minutes at 16,000 g at 4°C. The supernatant was then removed taking care not to disturb the pellet. The

pellet was washed in 250|l i1of 70% ethanol and centrifuged at the same speed and

temperature for 5 minutes. The supernatant was removed and the pellet vacuum- dried for 30 minutes before being stored overnight at -20°C. Pellets were suspended

to 4pi of RNA sample loading buffer (Sigma), heated to 95°C for 10 minutes and

cooled immediately on ice. Sequences were loaded onto a gel prepared by Dr J Sainta-Maria or Miss CTL Tran, and read by an ABI Prism 377 DNA Sequencer. Sequences were checked visually against the expected nucleotide sequence for the appropriate mRNA.