What are the steps of a molecular beacon RT-PCR assay?

There are five basic steps in molecular beacon RT-PCR assay. These steps are target design, primer design, optimization of amplification reaction (using SYBR Green), molecular beacon design, and molecular beacon synthesis, in that order respectively. The steps in the protocol are described below. 

1. Target design:  One should create primers which amplify a target region of 75 to 250 base pairs. One should avoid using a molecular beacon target sequence which forms strong secondary structures. 
2. Primer design: One should generate primers with a 50 to 60% GC content. The target primer melting temperature should be between 50 to 65 degrees celsius. One should also avoid repeats of Gs or Cs longer than three bases and place them on the ends of the primers. 
3. Using lower primer concentration decreases accumulation of primer-dimer formation and nonspecific product formation; this is crucial when optimizing the amplification reaction using SYBR Green I dye. The dye binds to all dsDNA and allows detection and monitoring by measuring the increase in fluorescence throughout the PCR cycle. 
4. Molecular beacons should be designed in a way that they are able to hybridize to their targets at the annealing temperature of the PCR. In the presence of a complementary target the molecular beacon must form a stable probe-target hybrid. If there is a mismatched target the MB must remain closed. Typically a probe between 22 to 30 nucleotides is used but may be expanded from 18 to 30 bases. The annealing temperature of the probe should be between 7 to 10 degrees celsius above the annealing temperature of the PCR.   
5. The molecular beacon can then be chemically synthesized by using standard automated synthesis methods. Dabcyl is typically first used at the 3’ end of the oligonucleotide. Then at the 5’ end of the oligonucleotide, an amino modifier or a thiol is applied for subsequent coupling to a fluorophore. Conversely, a fluorophore can also be directly applied using a phosphoramidite. After precipitating the molecular beacon with ethanol and salt and dissolving the pellet in a 100 microliter TE buffer, the yield and absorbance can be measured.