Size fractionation of total RNA by electrophoresis though formaldehyde gels enabled the specificity of our probes to be assessed. mRNA represents only 1-5% of total RNA in mammahan cells (Sambrook et al 1989) and Northern hybridisation can detect abundant mRNAs (0.1% or more of the mRNA population) using 10-20 pg of total RNA (Sambrook et al 1989). For detection of rare mRNAs either poly(A)"""RNA should be examined or more sensitive techniques such as RNA slot or dot blotting should be employed. Northern blotting enabled us to determine the exact treatments required for the filters to maintain specific signal but lose non-specific probe binding.
Gel electrophoresis
A formaldehyde containing 1% agarose gel is prepared by melting 0.775 g of agarose (Ultrapure, BRL, Life Technologies Inc, USA) in 73.3 ml of 1 x MOPS in a microwave oven. After allowing cooling to 60°C, 4.17 ml of 37% formaldehyde is added and gently mixed. The gel is then poured into a midigel box (Sa-PHOR horizontal gel electrophoresis system, Scotlab, Scotland) in a chemical hood and allowed to set for 30 minutes before being submerged in 1 x MOPS gel running buffer. 20 pg ahquots of RNA at 1 pg/pl in DEPC-treated water are removed and dried on a rotary evaporator (SpeedVac Concentrator, Stratech Scientific, London) for 30 minutes. The dried samples are resuspended in 25 pi of Northern gel loading buffer and incubated at 55 °C for 15 minutes in a water bath. After chilling on ice and centrifugation for 5 seconds to deposit all of the fluid in the bottom of the microfuge tubes 5 pi of Northern gel loading dye is added and the samples loaded into the gel lanes. The samples are run into the gel at 60V followed by electrophoresis at 25V until the bromophenol blue has migrated 10 cm (approximately 16 hours). During the run the gel running buffer is constantly recirculated using a pump. The gel is then stained with ethidium bromide (5 pg/ml in distilled water) for 5 minutes, destained by washing twice in distilled water for 10 minutes and photographed under UV illumination using a Polaroid DS34 camera with a DS H-13 hood (Genetic Research Instrumentation Ltd, Essex; Plate 5.2). The distances travelled by the 28S and 18S ribosomal RNA bands which represent 80-85% of total RNA (Sambrook et al 1989) are measured and used as reference molecular weight markers. The gel is then finally washed in 10 x SSC for
10 minutes at RTP prior to transfer to a nylon membrane.
Transfer o f RNA to nylon membranes
The RNA is transferred to a nylon membrane using capillary elution (Sambrook et al 1989). A standard blot apparatus is set up as shown in Figure 5.2.
A glass dish is partially filled with 10 x SSC and a glass plate is placed across the top of 4 rubber supports. 2 large pieces of 3 mm Whatman's chromatography paper
(Whatman, Maidstone) soaked in 10 x SSC are placed on the glass plate with the ends entering the reservoir of SSC to act as wicks. The gel is placed face down on the paper with the portion containing the wells removed.
A nylon filter (Hybond-N, Amersham, UK) soaked in 10 x SSC followed by three 3 mm Whatman's chromatography paper all cut exactly to the size of the gel are then placed on the gel. At each stage any air bubbles are removed using a glass rod. A stack of paper towels approximately 10 cm thick is then applied and weighted down with a glass plate and a 500 g weight. Saran Wrap is appHed to the large pieces of Whatman's paper around the gel to prevent bypassing of the SSC solution into the papers above.
The transfer is left overnight (or for a minimum of 12 hours) after which the nylon filter is removed and fixed.
Fixation o f RNA to nylon filters
The RNA is fixed by baking the nylon filter at 80°C for 10 minutes followed by exposure to UV light at 302 nm for 5 minutes. The filter can be used immediately in hybridisation reactions or can be stored at 4°C wrapped in Saran Wrap.
Hybridisation and autoradiography
In order to minimise non-specific and background staining the filters are prehybridised for 2 hours at 45°C using 10 ml of Northern hybridisation solution in a hybridisation oven (Techne Hybridiser HB-ID, Scotlab, UK). Following removal of hybridisation solution, 10 ml of fresh hybridisation solution containing radioactive labelled probe at 5 x 10^ dpm/ml is added and then incubated at 45 °C overnight (or for a minimum of 16 hours).
After hybridisation the filters were washed at low stringency using 2 x SSC with 0.1% SDS twice for 30 minutes at 45°C. The filters were then washed at high stringency using either 0.2 x SSC/0. 1% SDS or 0.1 x SSC/0.1% SDS for varying times at between 45 °C and 65 °C to assess the optimal requirement for each probe which
retained specific signal but lost non-specific and background signal.
Following the stringency washes autoradiographs were established by exposing the filter to X-ray film (Fuji Medical X-ray Film, Fuji Photo Film Company Ltd., Japan) at — 70°C with an intensifying screen for varying lengths of time. The autoradiographs were developed on an autoradiograph developer (Gevamatic 60, Agfa-Gevaert, Germany).
Filter wash and reprobing
Probed filters were washed by pouring 0.1% SDS solution at 100°C onto the filter and allowing to cool to room temperature on an orbital shaker (The Belly Dancer, Scotlab, UK). The filters were exposed to X-ray film at — 70®C to ensure complete probe removal prior to storage at 4°C or reprobing.