4.4.1 Preparation of solutions and equipm ent
RNA is prone to ribonuclease (RNase) digestion throughout extraction, purification, blotting and hybridisation. As a result, gloves were worn at all times and the working area kept scrupulously clean. All solutions used for RNA were prepared with sterile deionised water and made where possible from reagents kept separate from the general chemicals in the laboratory. Handling of reagents, solutions and tissues was kept to a minimum. Pipettes, tips, Eppendorf tubes and a midi-gel tank were reserved for use with RNA samples only. All tweezers, spatulas and homogenisers were rinsed with water, cleaned with ethanol and baked overnight at 200°C before use. Non-sterile plastic-ware such as the gel tank and combs were treated with RNase Free (CLP) and rinsed with sterile deionised water immediately prior to use. Sterile plastic-ware such as tips and tubes were assumed to be RNase free.
4.4.2 E xtraction of total RNA
4.4.2.1 Fetal tissue
Total RNA was isolated from fetal tissues using an adaptation of the guanidine isothiocyanate technique described by Chirgwin et ah (1979). Tissue samples were placed
Chapter 4. Methods
on dry ice and, while still frozen, dissected into aliquots of 100 mg or less using a scalpel blade. Each aliquot was then homogenised in 700 |Lil solution D. Samples were transferred
to Eppendorf tubes and mixed well with 90 |il 2 M sodium acetate (pH 4.0), 600 |xl water saturated phenol and 150 |il chloroform. After incubating for 15 minutes on ice, they were spun at 13,000 rpm for 10 minutes at 4°C. The upper aqueous phase (600 |xl) was removed to a fresh tube and mixed with 200 |il solution D, 20 |il 2 M sodium acetate, 500 |il phenol and 120 |il chloroform. Samples were then spun again at 13,000 rpm for 10 minutes at 4°C. The upper aqueous phase (400 p-l) was removed to a clean tube and 500 |xl isopropanol added. Samples were left at -70°C for at least 1 hour or overnight at -20°C.
RNA pellets were obtained by spinning at 13,000 rpm for 15 minutes at 4°C. Pellets were washed in 1 ml 70% ethanol, spun again briefly and left to air dry for 5-10 minutes. RNA was resuspended in 10-20 pi 1 mM EDTA. The concentration of RNA was determined spectrophotometrically as described for DNA (section 4.2.5) except that for RNA one OD unit = 40 pg/ml. Samples were stored at -70°C until use.
4.4.2.2 Cell pellets
Total RNA was extracted from cell pellets using a similar procedure to that described for fetal tissue. The total number of cells in suspension was estimated using a cell counting chamber prior to pellet formation. Seven hundred microlitres of solution D were used per 10^ cells.
4.4.3 R everse transcriptase polym erase chain reaction (RT-PCR)
Reverse transcription of RNA was used to generate cDNA for use in PCR amplification. Total RNA samples (1 pg diluted to 10.25 pi in water) were denatured at 70°C for 5 minutes and cooled to 37°C. Reverse transcription was carried out using 0.2 pg random hexanucleotide primers, Ix reverse transcriptase buffer, 10 mM dithiothreitol, 1 mM each dNTP, 40 units M-MLV reverse transcriptase (Gibco BRL) and 1 unit RNase inhibitor (Promega). These were added to a final volume of 20 pi and incubated at 37°C for 60 minutes. The reaction was then terminated by heating to 90°C for 5 minutes. Samples
were diluted with 80 |ll1 water and stored at -70°C until needed. PCR was performed (as described in section 4.2.12) using 1/20 volume (5 |il) of the cDNA template.
4.4.4 E lectrophoresis of RNA
The method used was adapted from those of Lehrach et al, (1977) and Goldberg (1980). RNA is heated to 65°C to remove any secondary structure which might interfere with the mobility of the RNA. Formaldehyde, used in the sample buffer and agarose gel, acts as a dénaturant to prevent secondary structures reforming during electrophoresis.
A 1.5% agarose gel was made up in Ix morpholinopropanesulphonic acid (MOPS) buffer and 2.2 M formaldehyde in a final volume of 400 ml. As formaldehyde is toxic, the gel was poured in the fume hood and left to cool. The running buffer used was Ix MOPS. Total RNA samples (10-60 pg in 15 pi volume) were mixed with 30 pi sample buffer, denatured at 65°C for 6 minutes and then quenched on ice for 2 minutes. Prior to loading on the gel, 1.5 pi 1 mg/ml ethidium bromide was added to each sample. Samples were run at 100 V for 2-4 hours or 30 V overnight. Separated RNA was visualised and photographed under UV light.
4.4.5 N orthern blotting
Size fractionated RNA species were transferred to nylon membrane using an adaptation of the Southern blotting technique (section 4.2.9). The method differed in the following respects: the gel was not denatured prior to blotting; the gel was inverted before transfer; and lOx SSC was used as the transfer buffer. Following blotting, membranes were baked for 2 hours at 80°C to permanently bind the RNA.
Chapter 4. Methods
4.4.6 N orthern hybridisation
The probes used for hybridisation with Northern blots were radiolabelled with [a-^^P]dCTP and separated from unincorporated nucleotides using identical methods to those used for Southern hybridisation (Sections 4.2.10.1 and 4.2.10.2).
4.4.6.1 Prehybridisation
Membranes were placed in sealed plasic bags or bottles containing 10 ml of Northern prehybridisation mix and an equal volume of formamide. Denatured salmon sperm DNA and formamide were added just before use and the mix pre-warmed. Membranes were incubated for a minimum of 2 hours at 42°C.
4.4.6.2 H ybridisation
The hybridisation mix was identical to that used for prehybridisation. Membranes were transferred to fresh bags. Probes were heated to 100°C for 8 minutes, quenched on ice and added to pre-warmed hybridisation mix. Membranes and mix were sealed in the bag and incubated overnight at 42°C.