Selection of RT-PCR

[aftabac]

HI
which type of RT-PCR is better for experimentation if we consider economically and also efficiency of performance.
1- With SYBR green dye
2-FRET-RT-PCR

what are the advantages and disadvantages of these PCR.

Waiting for answer
aftab

[oBWhat]

SYBR green dye seems to be the standard for RT-PCR. I think FRET is quite new so I would stick with SYBR

[katherine_ft]

I would also choose SYBR green dye.

The advantages of the SYBR Green dye chemistry are as follows:
• It can be used to monitor the amplification of any double-stranded DNA sequence.
• No probe is required, which reduces assay setup and running costs.

Disadvantage of SYBR Green I Dye
The primary disadvantage of the SYBR Green I dye chemistry is that it may generate false positive signals; i.e., because the SYBR Green dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences.

[danfive]

SYBR green can work but you must optimize so that no nonspecific products are generated---optimize using traditional PCR. Then a simple primer concentration optimization--usually reduction in primer used in traditional PCR--plus Melting point determination. You must also have a positive control reaction that works without generating nonspecific products---this control will give you a melting point (pcr amplicon's) that you can compare in your unknowns. That way if the melting points are the same---you can conclude that the SYBR green expression signal is all due to the one pcr product--and not to any primer dimer or nonspecific bands.