实时定量RT

I. Assay Development
A. Sequence selection
B. primer & Probe Selection
C. Quencher dye and inteRNAl reference
D. Assay Validation
II. Assay Setup
A. One- vs Two-Step RT-PCR
B. Plate Setups for Robotics
C. thermocycler settings
III. Data Analysis
A. Baseline and threshold settings
B. Standard Curves
C. Inter- vs intra-assay variABIlity
D. Sample normalization

taq Exonuclease Reaction

Sequence Selection
The most important component of building an assay
Make sure the initial sequence is the correct one
Use Blast at the ncbi to find other sequences (http://www.ncbi.nlm.nih.gov/)
Download all the unique sequences you find, including other species (human, mouse, rat)
Look particularly for the NM_ or refseqs