ROX reference dye for quantitative RT-PCR - (Sep/23/2011 )

Hi out there,

I am absolutely new in RT-PCR and have the following problem:
we have used a SYBR based quant. RT-PCR kit and had quite good results. We have now tried another kit from another company that also includes ROX as a reference dye. Despite running the RT-PCR on the same sample, we got different results. The detection of our target started at 14-15 cycles with the old kit and at 17-18 cycles with the new kit.
Also, that we have some issue with primer dimerization in the new kit although we are using the same primers as with the old kit.

here my questions:
1. what exactly is the reference dye there for?
2. is it possible that it interferes with the PCR and could it be responsible for the difference in the results? Or is it just due to the different test kit with different rev transcriptase etc.?


Any help is very much appreciated!!!!

Thanks in advance

Sabeene

Hi,
basically a reference dye like ROX is used to normalize the fluorescent reporter for any inaccuracies resulting from pipetting errors for example.
If you (by mistake) add more master mix than usual than your overall fluorescence for this reaction will be higher, but your ROX level will be higher as well what still allows you to compare your reaction with others including less master mix (and hence lower fluorescence).

The new kit could contain the ingredients in a different composition and hence producing different levels of amplification and fluorescence.

Please read the following regarding ROX:
Real-time PCR: Understanding Ct