technical limitations of the method
MDM2 Loading
Control 1 - CN m Q. a a Q Q Q g g g • • • MDM2 Loading Control
F ig u re 3-3 cDNA RDA detection of MDM2 in primary mouse embryonic fibroblasts 4 hours after a dose of 4 Gy of ionising irradiation. The representation blot (left panel) shows the up-regulation of MDM2 in irradiated tester relative to unirradiated driver populations. The difference products blot (right panel) depicts the pattern of enrichment of MDM2 in subsequent rounds of RU subtractions.
The RU final difference products were further characterised to investigate how the degree of up-regulation in the tester relative to the driver relates to the pattern of enrichment in the subsequent rounds of subtraction in cDNA RDA. Three gene fragments (R U l, RU2 and RU3) identified in RU DP3 were probed against separate representation blots and difference blots (figure 3-4). The representation blots confirm that these genes are all up-regulated in the irradiated tester relative to the unirradiated driver representations. The representation blots dem onstrated that the RU2 and RU3 gene fragment are up-regulated to a high degree in the tester whereas the R U l gene fragment, similarly to the MDM2 gene fragment (figure 3-3) displayed a lower degree of approximately 3-5 fold up-regulation following irradiation in the irradiated tester relative the unirradiated driver. The enrichment of R U l, RU2 and RU3 in the subsequent rounds of the RU subtractions was then examined by probing these gene fragments against separate RU difference products blots (figure 3-4). The enrichment patterns by the RU 1, RU2 and RU3 gene fragments was found to correlate with the relative degree of up-regulation in tester
compared to driver, suggesting that the higher the degree of up-regulation in a tester population the higher the degree of enrichment in subsequent rounds of subtraction. As a control, the XPB gene was probed against the blots. XPB was expressed at comparable levels in unirradiated and irradiated MEFs, and, as a result, it was not enriched in the successive rounds of subtraction.
RU REPRESENTATION BLOTS RU DIFFERENCE PRODUCTS BLOTS
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UL UL Q a o lU UJ 3 3 3 s s CE (£ OC m ^ R U 1 RUl — RU2 RU2 « RU3 RU3 XPB XPB Loading Loading Control ControlFigure 3-4 Enrichment patterns in subsequent rounds o f subtraction during the RU
cDNA RDA experiment of the R U l, RU2 and RU3 gene fragments. A gene fragment representing the XPB gene was probed against the representation blot and difference products blot as a control.
3.4
Statistical analysis of cDNA RDA
The subtraction described above was part of a joint project with Jenny Regan at the Trafford Centre for Medical Research at the University of Sussex. Ms. Regan performed a statistical analysis on randomly selected mammalian full-length cDNAs that were picked from the Genbank database (summarised in figure 3-5). The analysis
demonstrated that 8 6% of the genes investigated generate DpnII fragments that are in the amplifiable range of 1(X)-1200 bp. Therefore, these results imply that 8 6% of genes in a transcriptome will be present in a representation. This means that the theoretical
RDA experiment is p = 0.14, p = 0.02 and p = 0.003, respectively. The majority of the genes that did not generate amplifiable fragments are below 1 kb including the 3’ and 5’ untranslated regions. Amplifiable Range 1 0 0 -1 2 0 0 bp «2 250 250 500 750 1000 1250 Fragment Length (bp)
1
25 E O) 20 S. 15* S. n 10 g 5 Q . E < 2000 4000 6000 8000 10000 cDNA Length (bp) Number of g e n e s analysed: 200 Mean length of each gene: 2788 bp Dpnll digestMean fragment length: 286 bp Mean fragments/gene: 7.31
Mean amplifiable fragments/gene: 4.22
Percentage amplifiable fragments per digest: 57.7% Number of g e n e s that generate amplifiable fragments: 86 %
Figure 3-5 Statistical analysis of the theoretical limitations of cDNA RDA by Jenny
Regan (TCMR, University of Sussex, UK). 200 genes were analysed. The upper graph
depicts the relationship between the num ber o f fragments generated by a DpnW digest
and their size distribution. The lower graph shows the relationship between transcript
size and number of am plifiable fragm ents generated by the DpnW digest.
3.5
D iscussion
Ionising irradiation activates the DNA damage response pathway, which leads to up- regulation of transcription of genes such as MDM2, whose products facilitate the cellular responses to DNA damage (reviewed in Durocher, et al., 2001 ; Khanna, et al., 2001;
Zhou, et al., 2000b). The results presented in this chapter demonstrate that cDNA RDA is able to detect up-regulation o f transcripts in primary mouse embryonic fibroblasts 4 hours after treating the cells with 4 Gy o f ionising irradiation.
The results from the RU experiment provide some insights into the sensitivity of cDNA RDA. While the MDM2 and R U l gene fragments were clearly detected in the final difference products of RU, they did not enrich to the same high degree as the RU2 and RU3 fragments with increasing stringencies during successive rounds of subtraction. Therefore, these findings suggest that the higher the degree of up-regulation of the transcripts in the tester population relative to the driver population, the higher the enrichment of gene fragments representing this transcript in successive rounds of
subtraction during cDNA RDA. Both MDM2 and R U l were detected in this experiments demonstrating that cDNA RDA is capable of detecting low degree of up-regulations. However, the implication of these observations is that it is questionable whether transcripts that are up-regulated to very low degrees in the tester population will be detected in the final difference products when high ratios between tester and driver are used.
Although cDNA RDA has been successfully employed by several groups to identify differential gene transcription, the technical limitations o f the technique have not been clear (Hubank, et at., 1994; Cohen-Salmon, et at., 1997; Gress, et al., 1997; Lewis, et al.,
1997; Zheng, et al., 1997; Melia, et al., 1998; Bakin, et al., 1999; Ouyang, et al., 1999). The statistical analysis carried out by Jenny Regan demonstrate that cDNA RDA is capable of detecting genes with a theoretical probability of p = 0.86. From this it follows that the theoretical probability o f cDNA RDA of failing to detect two or more transcripts that are part of a given transcriptome is p < 0.0 2.
In summary, the experiments described here demonstrate that cDNA RDA is a sensitive method that is capable of detecting transcripts that are up-regulated at least 3-5 fold, 4 hours following treatment with 4 Gy o f ionising irradiation. The statistical analysis shows that cDNA RDA is very sensitive and theoretically capable of detecting, with high probability, the vast majority of differential gene transcription between two populations. Therefore, cDNA RDA is an appropriate method to investigate whether Ku, DNA-PKcs or the active DNA-PK complex modulates gene transcription of genes whose altered transcription is required for, or facilitates NHEJ repair.