ZONA SUCIA

2.15 PREPARATION OF SOLUTIONS, GLASSWARE AND

PLASTICWARE FOR RNA WORK

Gloves were worn at all times and efforts were made to assure that the working area was dust free. All solutions for RNA work were made, where possible, from solids which were kept separate from the general chemicals in the laboratory. These solids were weighed out using sterile plasticware. Wherever possible, solutions were treated with 0.05% diethyl pyrocarbonate (DEPC), left overnight at room temperature, then

autoclaved. DEPC is a potent inhibitor of ribonuclease (RNAse) and is broken down to ethanol and CO2 by autoclaving (Ehrenberg et a l, 1976). Solutions containing Tris or

EDTA were not DEPC treated, nor were solutions which could not be autoclaved (such as SDS). These solutions were made up in DEPC-treated distilled deionised water, in bottles which had been DEPC-treated and autoclaved. Glassware was baked overnight at 180°C before use, while sterile plasticware was assumed to be RNAse-free.

2.16 EXTRACTION OF TOTAL RNA FROM MOUSE

EMBRYOS

Total RNA was extracted from mouse embryos using TRIzol Reagent (Gibco BRL), which is based on the guanidine isothiocyanate/phenol method developed by

Chomczynski and Sacchi (1987). TRIzol promotes formation of RNA complexes with guanidine and water molecules and inhibits hydrophilic interactions of DNA and proteins. As a consequence, DNA and proteins are excluded from the aqueous phase, leaving the RNA which can then be purified. The RNA produced is of high quality and contains the whole range of cellular RNA molecules.

Embryos were homogenised in TRIzol by repeated sucking up and down through a needle, using a disposable syringe. The gauge of the needle was gradually decreased, from 25G to 19G, until a fine suspension was formed. The volume of TRIzol used depended on the embryo age. Single E8.5 embryos were homogenised in 200 pi volumes, E9.5 in 500 pi, E10.5 in 1 ml, El 1.5 day in 2 ml and E12.5 in 3 ml. The homogenised samples were incubated at room temperature for 5 minutes to complete the dissociation of nucleoprotein complexes, then spun for 5 minutes at 13,000 rpm to pellet cellular debris. The supernatant was decanted into a clean tube, and one fifth volume chloroform was added. The samples were shaken vigorously by hand for 15 seconds then incubated at room temperature for 2-3 minutes. Phase separation was achieved by centrifugation at 13,000 rpm for 15 minutes at 4°C. The colourless upper aqueous phase contains the RNA, and this was removed into a clean eppendorf, taking care not to take any of the protein-containing interface or the lower red, phenol-chloroform phase containing the DNA and proteins. The aqueous phase was mixed with an equal volume of isopropanol, then left to stand at room temperature for 10 minutes to precipitate the RNA. The samples were again centrifuged at 13,000 rpm at 4°C for 15 minutes, to pellet the RNA. The supernatant was discarded and the pellet washed in 75% ethanol before respinning at the same speed for 5 minutes. The pellet was air dried and

resuspended in 10-300 pi DEPC ddH20, depending on the size of the pellet. The RNA was stored at -70°C or -20°C until use.

The mass of RNA extracted from individual embryos was assumed to be approximately 1 pg at E8.5, 2 pg at E9.5, 20 pg at E10.5 and 50 pg at E12.5 (extrapolated from data given by Estibeiro et a l, 1990).

2.17 REVERSE TRANSCRIPTION (RT)-PCR

RT-PCR was used to analyse gene expression during embryonic development, and to generate cDNA fragments for sequence analysis. Total RNA was used as a template for first strand cDNA synthesis, by reverse transcription, and this single stranded cDNA was then used as a template for PCR.

2.17.1 Reverse Transcription

Reverse transcription was performed routinely from total RNA and using random hexanucleotides as the primers. Hexanucleotides were annealed to the RNA by mixing together approximately 300-500 ng total RNA (for example, half the total RNA

extracted from a single E8.5 embryo) and 0.2 pg random hexanucleotides (Gibco BRL) in a volume of 9.5 pi. The mixture was denatured by heating to 70°C for 6-7 minutes, then annealed by incubating at 37°C for 10 minutes.

The reverse transcription reaction was performed in a 20 pi volume containing the 9.5 pi annealed RNA/hexanucleotide mixture, 1 x first strand RT reaction buffer, 0.25 mM each dNTP, 10 mM DTT, 0.5 pi RNase inhibitor (Pharmacia) and 1 pi (200 Units) Moloney murine leukemia virus (MMLV) reverse transcriptase (Gibco BRL). The RT mix was prepared as a single batch for each set of reactions, and then aliquoted. Each RNA sample was also used to prepare a “No-RT” control; the annealing step was performed as described above, and a reaction mix was set up containing all the components above except the reverse transcriptase. RT and No-RT reactions were incubated at 37°C for 1 hour, then stopped by heating to 95°C for 5 minutes. The first strand cDNA product was then diluted to 40 pi and stored at -20°C.

2.17.2 PCR

RT-PCR was carried out as described for the conventional PCR reactions (section 2.4), using 1 pi first strand cDNA product. For the purposes of gene expression analysis, primers were designed, wherever possible, to flank an intron. PCR was also carried out with the “No-RT” control samples, to verify that the PCR is priming off the cDNA and not off of contaminating genomic DNA. Control PCR reactions were carried out with primers specific for hypoxathine-guanine phosphoribosyltransferase (HPRT), a ubiquitously expressed gene (Melton et al, 1984).