Qiagen RT-PCR Kit (Rotor-Gene Q)
Posted 01 June 2011 - 05:55 AM
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
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.
Posted 06 June 2011 - 04:32 AM
Multiplex is optimised for running two or more sets of primers and hydrolysis probes (different probe dyes) in the same reaction. Unless you don't do multiplex, you probably won't need it.
Probe mix is for use with hydrolysis probes, if you use probes you need to pick this one (or the Multiplex).
SYBR uses intercalating dyes and requires only primers to run. It's a completely different method of quantification.
You need to decide if you are going to design and use probes or SYBR, probes are more expensive but usually more specific, and you can multiplex them. SYBR is cheaper and you can do melting at the end to check specifity and dimers. My guess is you only have the primers, so you need to do SYBR method, but find some info about it first, it's very basic.
Multiplex reaction (two or more genes in the same reaction, but you can't do this with SYBR, only with differently labeled probes) is always more difficult to optimise than singleplex, so I wouldn't recomended to a beginner, only if you have low amount of template you might consider it. Every single reaction must be optimised separately and then again together, some product may be more abundant, so you have to decrease (limit) primers for that gene.
I never trust anything that can't be doubted.
Posted 06 June 2011 - 08:15 PM
I appreciate your kindness. I would certainly bug you from time to time for guidance. God bless you.