2.12.1 Working with RNA
RNA is very sensitive to degradation RNAses which are present in the environment. For this reason all reagents used were prepared in diethylpyrocarbonate (DEPC) treated MilliQ water, or solutions were DEPC treated when made up. This was prepared by adding 1ml of DE PC/litre of water or prepared solution, and incubating at 37°C for at least 4 hours, before autoclaving to remove residual DEPC. This treatment destroys RNAses. Wherever possible, equipment such as gel apparatus and boxes for incubations were used for RNA work only, or were rinsed with a dilute solution of hydrogen peroxide followed by DEPC treated water before use. Sterile plasticware, which is RNAse free, was used wherever possible for the preparation of solutions.
2.12.2 Extraction of RNA
For the purification of sufficient mRNA for northern blotting, a 25cm^ flask of fibroblasts was used for each stimuli or time point measured. Cells were infected in the usual way, or treated with cytokines as detailed in the appropriate experiments. RNA was purified using RNAzol B™ (Biotecx Laboratories, Houston, Texas), a method which reduces the guanidium isothyocyanate extraction and phenol chloroform extraction to a single step. The method used was essentially as described in the protocol supplied by the manufacturers. Cells were homogenised with RNAzol B solution before addition of chloroform and centrifugation to separate the phenol chloroform and aqueous phases. The aqueous phase containing the RNA was added to isopropanol and centrifuged, resulting in the pelleting of the precipitated RNA. The pellet was washed in 75% ethanol and dried under vacuum, before resuspension in DEPC treated water. The absorbance at 260 and 280 nm was measured to determine the amount of RNA present and its purity. A 260:280 ratio of 2 indicates pure RNA, free of protein contamination. The amount of RNA present in the sample (in mg) was calculated from the formula:
2.12.3 Denaturing gel electrophoresis
A 1% agarose gel was made by combining 3g agarose with 230 mis DEPC water. When the agarose was melted, 15 ml 20X MOPS running buffer (0.4M MOPS, 0.02M EDTA, 0.2M sodium acetate, pH 7), and 54 ml formaldehyde solution were added. The samples were adjusted so that between 7.5 and lOpg RNA was loaded per lane of the gel, following dilution in RNA sample buffer (7pl formaldehyde solution, 4pl 5X MOPS running buffer, 2pl ethidium bromide solution from 1 mg/ml stock, 20pl form amide, 2.5pl bromophenol blue). The gel was run using precooled IX MOPS, at 130 volts until the bromophenol blue had migrated about 3 cm. The gel was then observed under UV light, and the 28S and 18S bands could be seen. The gel was photographed to demonstrate equal loading of samples. (Polaroid CU5 88-46 Land Camera). The gel was then agitated in DEPC treated water for 30 minutes to remove formaldehyde.
2.12.4 Transfer of RNA to membranes
The RNA was transferred, overnight, to the nylon membrane (Boehringer Mannheim, Lewes, East Sussex, UK) by capillary action. The gel apparatus was used as a blotting apparatus, with the reservoirs filled with 20X SSC (3M Sodium Chloride, 0.3M Sodium citrate, pH 7). The RNA was fixed to the membrane by baking at 120°C for 20 minutes. The blot was then cut into pieces for hybridising with individual probes, and sealed into hybridisation bags.
2.12.5 Principles of the digoxigenin (DIG) detection system.
The DIG detection system is based on the steroid hapten digoxigenin, which is a plant product and thus avoids problems with background activity which are encountered with other haptens. DIG labelled DNA probes are hybridised to mRNA which is immobilised on a nylon membrane during northern blotting. The hybrids are detected with an anti-DIG antibody (Fab fragment) conjugated to alkaline phosphatase. The signal is detected using a chemiluminescent substrate, CSPD®, followed by autoradiography. The DIG system was chosen because of the safety of the technique in comparison to radioactive detection, and the equally high sensitivity.
2.12.6 Hybridisation with DIG-labelled oligonucleotides
The following reagents were used for the hybridisation stages:
• Buffer 1:- 0.1M maleic acid, 0.15M sodium chloride, adjusted to pH 7.5 with solid sodium hydroxide)
• Blocking stock solution:- lOg Boehringer Mannheim blocking reagent, 100ml buffer 1, heated to dissolve.
• Buffer 2 - Blocking stock solution diluted 1:10 in buffer 1
• Buffer 3 - 0.1M Tris, 0.1M sodium chloride, 50mM magnesium chloride, adjusted to pH 9.5 with hydrochloric acid.
• Hybridisation solution - 5X SSC (0.75M sodium chloride, 0.075M sodium citrate, pH 7.0; DEPC treated), 1% blocking reagent (1:10 dilution from stock), 0.1% Sarkosyl, 0.02% sodium dodecyl sulphate (SDS).
The membrane was prehybridised by incubation at 42°C for 1 hour in hybridisation solution. The solution was replaced with the DIG-labelled DNA probe for the appropriate chemokine in the same buffer, and hybridised overnight at 42°C. The blot was then washed in 2X SSC + 0.1% SDS for 2 x 5 minutes, and in 0.1 X SSC + 0.1% SDS for 2 x 5 minutes. All steps were carried out at 42°C.
2.12.7 DIG labelled probes.
DIG-labelled DNA probes (30 or 31-mer) specific for IL-8, MCP-1, RANTES, MIP-1a, and p-actin mRNA were used at lOng/ml (R&D Systems).
2.12.8 DIG luminescence detection.
This was carried out using components from the Boehringer Mannheim kit No. 1363524, essentially as detailed in the manufacturers instruction sheet. All steps were carried out at room temperature. The membrane was washed for 5 minutes in washing buffer (0.3% Tween-20 in buffer 1). The membrane was blocked for 30 minutes in buffer 2, then incubated for 30 minutes in anti-DIG antibody conjugated to alkaline phosphatase, diluted 1/10,000. The membrane was then washed in washing buffer for 2 x 15 minutes, then equilibrated for 5 minutes in buffer 3. The lumingen was then diluted 1/100 in buffer 3. The membrane was placed on a plastic sheet, and the substrate, CSPD®, added (1 ml/membrane). The membrane was covered with the plastic sheet, sealed to
make a bag, and incubated at 37°C for 15 minutes. This was then exposed to X-ray film for 1-2 hours. Further exposures were done depending on the initial exposure. The signal was seen as a dark band on the X-ray film.
Ethidium bromide was included in each sample, enabling confirmation of equal loading by densitometric analysis. IL- 8 specific bands on X-ray film were
quantified by densitometric analysis (Biorad imaging densitometer) using the Molecular Analyst program. Corrections were made for small differences in loading by comparison with the p-actin control.
Table 2.1 Murine monoclonal antibodies specific for CMV antigens
Specificity Clone Isotype Source
CMV IE antigens 1 and 2 E 13' lgGl Biosoft, Paris, France CMV IE antigens 1 63.27^ lgGl Dr. W. Britt, Alabama, USA.
glycoprotein B (gB) (CMV late antigen)
7.17’ lgG3 Dr. W. Britt, Alabama, USA.
^ Suitable for flow cytometry or immunofluorescent staining on slides. Not suitable for flow cytometry.
Table 2.2. Primary murine monoclonal antibodies used in this study^.
CD number
Alternative names
Clone Isotype Proposed
function
Main expression Source
class 1 HLA