Figure 3

PCR and dot blot methods to assay combination lengths and composition. All samples were amplified across the multiple cassette region via PCR and the products were separated with gel electrophoresis. All samples were also subject to a control G418 resistance gene (neo^r) amplification reaction to verify the integrity of the extracted sample (data not shown; all samples positive). The PCR products were immobilized onto membranes and probed using shRNA-specific probes to characterize the component shRNAs of each amplified product. This figure shows a representative example of (A) the raw PCR separations and (B) dot blot exposures for the first 96 6.3 populations amplified and probed for shRNAs #3 and #8. n.b. smaller products were poorly amplified with the reaction conditions optimized for longer products, making visualization sometimes difficult. Several samples had multiple bands (#20 - 4,3; #24-5,4; #35-6, 3; #90-4, 3; #91-6, 4), for most of which the larger size was more readily detected. These were scored as the largest size. CM: C assette M arker (a custom 1-6 cassette marker made by PCR of the plasmid stocks). M: size M arker (standard 100 bp and 1 kb DNA ladders, Invitrogen). Dot blots were scored qualitatively as detected (+ve)/not detected (-ve) above background levels, taking into account the presence/absence of PCR products detected by gel electrophoresis for weakly detected bands. Probe #9 bound the least efficiently; some weakly detected products seen on the original films may not be apparent in the reproduced images. Samples with disparate results between the two methods correlated with poorly amplified products that were difficult to visualize with electrophoresis and were consequently weakly detected by dot blot analysis (red dots).