3. Add to wells in the PCR plate
2ul of 800nM forward primer to A1-12
2ul of 600nM forward primer to B1-12
2ul of 500nM forward primer to C1-12
2ul of 400nM forward primer to D1-12
2ul of 300nM forward primer to E1-12
2ul of 200nM forward primer to F1-12
4. Add to wells in the PCR plate
2ul of 800nM reverse primer to 1, 2 A-F
2ul of 600nM reverse primer to 3,4 A-F
2ul of 500nM reverse primer to 5,6 A-F
2ul of 400nM reverse primer to 7,8 A-F
2ul of 300nM reverse primer to 9, 10 A-F
2ul of 200nM reverse primer to 11, 12 A-F
5. Add 16 ul of master mix to each tube
6. Set up the thermal cycling conditions for the ABI Prism 7700 sequence detector as described previously:
50℃, 2 mins
95℃, 10 mins
40 cycles of the following:
95℃, 15 sec
60℃, 1 min
7. Following amplification, select the concentration of primers that show a reduction in DRn, but little effect on CT.
N.B. If limiting primer concentrations cannot be defined, or if the internal standard is less abundant than the target gene, amplification of individual genes may be carried out in separate wells or primers should be redesigned.
e) Real-time quantitative PCR
Once each of the steps a-d have been carried out, the quantitative PCR reaction may then be performed.
1. Prepare a mastermix of the reaction mix below so that there is enough for all samples to be investigated.
2. Pipette 15 ul of the PCR mix above into individual wells on a 96-well plate
3. Add 5ul of each cDNA sample to separate wells in the plate.
4. Subject the plate to the following cycling on the ABI Prism 7700 sequence etector:
50°C, 2 mins
95°C, 10 mins
40 cycles of the following:
95°C, 15 sec
60°C, 1 min
5. Following amplification, compare CT values of samples (normalised to internal standard) in order to assess fold differences in mRNA levels of the target genes.
