Methods in Anopheles Research - MR4

8070Mutant (S1014)Chapter 8 : Field Techniques8.5 Real-Time PCR Assays8.5.1 Vector Population Monitoring Tool using Real-Time PCR8.5.1.3 Knockdown resistance (kdr) assaysPage 4 of 6Heterozygous60FAM fluorescence (kdr-e allele)50403020100-10-20No template-10 0Wild Type (L1014F)10 20 30 40 50 60 70 80VIC fluorescence (wild type allele)Figure 8.5.1.3.3. Scatter plot analysis of TaqMan fluorescence data In this example real time PCR wascarried out using the east kdr assay on ~70 samples then fluorescence values of the FAM labeled probespecific for the kdr-e mutation were plotted against the VIC labeled probe specific for the wild type allele.Notes and TroubleshootingIf separate runs have been performed on the same samples for kdr-e and kdr-w, when interpreting theresults it is useful to have two copies of the real-time PCR machine software open on the computer atonce. In one copy open the kdr-w run and in the other open the kdr-e run. The same sample can thenviewed in each assay by moving between the two runs/copies of the software. This helps to rapidly assigneach sample a genotype. For example, sample one is examined in the kdr-W run and shows a signal inthe FAM channel and not in the VIC channel and when examined in the kdr-E run shows a signal inneither channel. Sample one is therefore scored kdr-W homozygous. Sample two is examined in the kdr-W run and shows an intermediate signal in the VIC channel but no signal in the FAM channel, whenexamined in the kdr-E run it shows an intermediate signal in both the FAM and VIC channels. Sample twois therefore scored kdr-E heterozygous. Alternatively if the east and west assays were run together on thesame samples the same approach can be followed using a single copy of the software.In most of our experiments using wild-caught mosquito specimens, we have found that interpreting theresults of the kdr TaqMan assays is relatively straightforward. However in some instances when using‘quick and dirty’ DNA extraction protocols, we have sometimes seen background signals which have