system for ultimate use in the context of dissected mouse somites and presomitic mesoderm. Issues involving the reliable extraction and poly (A)+ enrichment of starting RNA, the reproducibility of differential display with minute amounts of material and efficient cloning of differential display products will be discussed. The initial findings of a comparison of whole mouse embryos at different developmental stages will be presented.
3.1 RNA preparation
A standard technique for the extraction of RNA from small amounts of tissue is the acid-guanidinium-phenol method (Chomczynski and Saatchi, 1987). It has been widely used in mouse embryology (Harrison et al, 1995) and in situations where RNA is being extracted from only a few cells (Brady and Iscove, 1993; Dulac and Axel, 1995). The method was adopted for these reasons. Other techniques were assessed for example the direct extraction of poly (A)+ RNA from cell lysates using oligo-dT cellulose (Sambrook et al, 1989) but these were rejected as being
unreliable.
An important disadvantage of the method is that with very small volumes, some genomic DNA contamination is experienced. Given that the screening technique was to be PCR based, it was decided to routinely poly (A)+ enrich all RNA prior to differential display. This had the added advantage of removing ribosomal RNA from the starting material. Oligo-dT coated latex beads (Oligotex-dT^^, Qiagen) were chosen for this purpose because the protocol required a single RNA pipetting step and could provide enriched RNA in volumes as low as 20pl. In general, total RNA
extracted from small amounts of tissue {e.g. dissected somites) was not precipitated but used directly for poly (A)+ enrichment. In control experiments, an estimated 3.5p.g enriched RNA could be extracted from lOOjLig total RNA using this method.
3.2. Differential display
3.2.1 Reagents
Initially, the conventional protocol of Liang and Pardee, 1992 was followed (refer to section 2.3.7). However one base anchored primers were used for reverse
transcription (Bauer et a l, 1993; Liang et al, 1994) and [a^^PJdATF was used for the PCR step (Bauer et a l, 1993; Liang et al, 1995). The steps in the method are
itemised below;
1. Reverse transcription was with a single base anchored dTi2NX
primer.
2. One tenth of the reverse transcription reaction was used per PCR. 3. PCR was performed with the reverse transcription primer plus any
one of a panel of arbitrarily chosen lOmer primers. 4. The PCR annealing temperature was 40°C throughout
A panel of arbitrarily chosen 10-mer oligonucleotides named G1 to 08 was generated by the NIMR Oligonucleotide Synthesis Service (refer to section 2.1.3).
3.2.2 Gel type
As discussed earlier, it has been well established that native polyacrylamide gels give equally good resolution of PCR products with similar numbers of band differences between starting materials when compared with denaturing gels (Bauer et a l, 1993; Liang et a l, 1995). Native gels were used in view of this and their relative ease of handling. Best results were to be had if the gels were pre - run for 30 minutes at 4°C and then run at 30V/cm for the desired length of time. The clearest autoradiographic results were achieved with Kodak^^ BIOMAX-MR X-ray film. Kodak™ X-OMAT AR and Fuji™ RX were also tried but generally gave inferior results.
3.2.3 The effect of polv (A)+ selection
Major concerns in differential display centre around the theoretical risks of
amplifying contaminant genomic or plasmid DNA species and reverse transcribed ribosomal RNA. It is possible that amplicons from either of these materials could be present as differential bands after the PCR. In order to address these issues,
differential display PCR reactions were performed using total and poly (A)+ enriched RNA from 13.5 dpc mouse embryos before and after reverse transcription.
Figure 2.1 shows the fingerprints obtained when 5)Xg total and 500ng poly (A)+ enriched RNA were used as templates for an arbitrarily primed PCR reaction of 40 cycles using 07 without prior reverse transcription. Clearly products are seen in both tracks but are much reduced after poly (A)+ enrichment. This indicates that
(presumably) genomic DNA contamination in the total RNA preparation is largely removed by poly (A)+ selection. It is of course possible that the total RNA preparation used here was contaminated with extraneous plasmid DNA from elsewhere in the laboratory and that this was eliminated by the enrichment process.
Figure 2.2 shows the fingerprints obtained for each of these templates after reverse transcription. As per the Liang and Pardee protocol, only one tenth of the first strand cDNA was used in the PCR reaction. The amount of starting material indicated above each track corresponds to the amount that was reverse transcribed. A radically
different pattern is seen for each with many more bands and a higher general background smear in each lane. This latter feature is probably because the reverse transcription primer is used in the PCR reaction and so, in principle at least, every RNA that is reverse transcribed is amplified at least asymmetrically {i.e. with one primer only) 40 times. Although the tracks share many common bands, poly (A)+ enrichment definitely removes species that are present in the total population.
Presumably, these represent ribosomal or other non - poly (A)+ RNA derived cDNA species.
It is not clear that the differential bands seen in figure 2.1 contribute at all to the patterns seen in figure 2.2. It is possible that the huge excess of cDNA template generated after reverse transcription reduces the probability of significant amplification of whatever small DNA contamination existed in the RNA
preparations. During the preparation of total RNA samples derived from very small amounts of tissue, where the extraction volumes are kept necessarily low, the genomic DNA contamination incurred due to pipetting errors is proportionately higher. For this reason alone, poly (A)+ selection is probably of benefit.
3.2.3 The effect of RNA concentration
The original differential display protocols utilized several hundred nanograms of RNA (Liang and Pardee, 1992). In order to assess the effect of reducing the amount of RNA used on the differential display fingerprint obtained for a given tissue, serial
Figure 2.1
Autoradiograph representing the reaction products of an arbitrarily primed PCR performed on 5|ig total and 500ng poly (A)+ enriched RNA from 13.5 dpc mouse embryos without prior reverse transcription. The primer used was 07 and standard differential display reaction conditions were applied. The products were separated by electrophoresis through a native 5% (w/v) polyacrylamide gel run at 50W for two hours at 4°C. All reaction products must be due to contaminating DNA of whatever origin and these are considerably diminished by poly (A)+ enrichment.