Real-time quantitative RT -PCR (Taqman)
This is performed as a 2-step reaction:
1. cDNA synthesis from DNase 1-treated total RNA
2. PCR
1. cDNA synthesis (Advantageä RT-for-PCR Kit - Clontech)
All reagents listed are provided with the kit
a) Quickly thaw each tube in kit and place on ice
b) Spin each tube briefly in a tabletop microcentrifuge and return to ice
c) In a sterile 0.5ml microcentrifuge tube, add purified DNase 1 -treated RNA preparation to a volume of DEPC-treated H2O that will give a total volume of 12.5 ul. (Can use 0.2-1ug of total RNA, but recommend 1ug where possible). Use the same amount of RNA for each sample.
d) Add 1ul of either the random hexamer or the oligo(dT)18 primer (both are provided with the kit). (Oligo(dT) priming is the method of choice; however, if the 5
