3 replies to this topic

[Jevandrix]

Hi everyone,

First, let me brief you on the background of my research is to determine the expression of known-immune genes in pathogen-challenged fish. The RT-PCR cycler available is Qiagen's Rotor-GeneQ. The optimized kit listed for gene expression at Qiagen website is as following for both one-step and two-step PCR:

1. Rotor-Gene Multiplex
2. Rotor-Gene Probe
3. Rotor-Gene SYBR Green

Questions:

1. What is the difference between each kits?

2. What are advantage and disadvantage of each kit and which is the standard kit normally used?

3. Which one is relevant for me, an RT-PCR beginner with new unoptimized designed primers for multiple genes? Should I go straight ahead and purchase Multiplex?

4. If I do purchase Multiplex kit, can I still optimize the primer through Singleplex and adjust the conditions (i.e. Annealing temperature) for Multiplex reaction?

I have already mailed these questions to the service provider. However, inputs from experienced users are invaluable. Thank you.

Much Obliged,
Jevandrix

[pcrman]

I would suggest you, as a beginner, to start with two-step RT-PCR and single gene assay. By doing two-step RT-PCR, you will have a better understanding of how RT-PCR works, and a better chance of troubleshooting potential problems, and also have cDNA for multiple PCR of many genes.

[Jevandrix]

pcrman, on 05 June 2011 - 08:40 AM, said:

I would suggest you, as a beginner, to start with two-step RT-PCR and single gene assay. By doing two-step RT-PCR, you will have a better understanding of how RT-PCR works, and a better chance of troubleshooting potential problems, and also have cDNA for multiple PCR of many genes.

Thank you for your reply. I do happen to be attracted to two-step and SYBR-Green kit. However, would you mind explaining to me the difference between probe and SYBR-green from the kit type?

Much Obliged,
Jeevan

[pcrman]

Using a probe such as TaqMan PCR is more specific than SYBR green because in Taqman only specific binding to a probe to the PCR products produces a signal, while in SYBR green PCR, any dsDNA can give a signal including primer dimers.