Reporter, Quencher and Internal Reference Dyes
1- The classical reporter dye is 6-FAM (fluorescein)
2- Other reporters used for multiplexing are Joe and Vic.
3- Some other real-time machines, such as the Stratagene Mx4000, can use red dyes as reporters as well
4- The classic quencher dye has been TAMRA (rhodomine)
5- Newer quenchers are the dark dyes, DABYCL and the black hole quenchers (Biosearch Technologies)
6- TAMRA-quenched probes do not require a reference dye;they can use the TAMRA itself
7- Single probe reactions quenched by dark dyes should use an internal reference dye, classically ROX (dark red)
8- Multiplex reactions usually use dark quenchers and ROX
Assay Validation
1- Test primer pairs in all combinations with the probe with a known template, eg., plasmid clone, sDNA, RNA
2- Use standard assay conditions: 300-400 nM primers;100 nM probe, 3 mM MgCl2
3- Choose the primer pair that gives the highest deltaRn and the lowest Ct
4- Make a dilution of a template, either sDNA, sRNA or total RNA for a standard curve
5- If the slope of the standard curve of the best primer pair is around -3.5 increase the MgCl2 to 5 mM
6- If the slope is higher than -3.6, find another primer
7- An ideal assay will have a slope of -3.3
Primer Combinations - hGAP43
One vs Two-Step RT-PCR
Early experiments comparing SSII RT and Taq Gold in one- and two-step RT-PCR showed two-step reactions were superior in detecting low abundance transcripts
New one-step kits may give better results
·One-step method
Easier and faster to setup
May be best method when only a few assays are run repeatedly
Costs are higher (kits) and sensitivity still an issue
