Hi, 1.Definately you can use qPCR primers for RT. You have to use only reverse primer for RT i.e. for synthesis of first strand of cDNA. 2. Before adding more than one primer you have to check the complementarities between these primers. There should be no dimer formation between these primers. Thes primers should not compete strongly with each other. In other words you have to find 'Limiting Primer Concentrations'. If you are setting multiplex qPCR then you must have done this background work already. Then plz forgive me for long suggestions!! Best luck.

If annealing temp of your primers is same, then you can use all these together. otherwise you have to run separate RT PCRs

Hi, 1.Definately you can use qPCR primers for RT. You have to use only reverse primer for RT i.e. for synthesis of first strand of cDNA. 2. Before adding more than one primer you have to check the complementarities between these primers. There should be no dimer formation between these primers. Thes primers should not compete strongly with each other. In other words you have to find 'Limiting Primer Concentrations'. If you are setting multiplex qPCR then you must have done this background work already. Then plz forgive me for long suggestions!! Best luck.

Thanks. much appreciated

The multiplex PCR have following things;

1. Tm of your primers should be same

2. Run separate primers for optimization for primer concentration and gradient Tm range

3. than run two primer togather with their optimized (probably approximately same Tm) Tm

4. than increase to 3 and 4 porimers for real time quantification

5. It is important to select Fluorochrome also with a spectrum difference for each label by approximately 15 - 20nm with approximately similar coefficient of extinction patterns

You can also run Reference gene such as GAPDH beta Actin or tubilin etc see proper amplification

Hope to enjoy real time quantification

thanks