realtime pcr validating the results of microarray? - (Sep/06/2005 )
For validating the results of microarray,I wanna do realtime-pcr,including
ten or so genes， and I can't decide which is better,syber Green or Taqmen? What do you usually choose?
Syb green is meant for first time users, but can have non specific signals. Taqman needs time to design the probe and it costs more.
Althought the taqman system costs more, it is much quicker. No optimising protocols or cloning vectors to use as standards. I prefer the taqman over the cybergreen methods.
I like SYBR green. As long as you run the appropriate negative controls (i.e., non-reverse transcriptase rxn, water alone), you don't need to worry too much about detection of nonspecific products. For good info here, check out BioRad. Good luck with whatever you decide.
generally its quicker and easier (less optimisation) to use taqman assays on demand - providing they already have your GOI. however it is expensive for 10 genes.
sybr is cheaper but takes more optimisation and design. If you have not done much real time and can afford to use the taqman assays i would use those.
altho on the other hand syb green isnt difficult to use once optimised
It also depends on your machine which method you chose. You can't use Syber green on a machine that doesnt run melt curves, because then there is no way you can check for non specific product.
u can use syb at first, designing you primers in a way a taqmanprobe can fit between....when u are ot sattisfied, just order the additional probe..
wih probes u can multiplex....that SAVES money...but it is some kind of investment
I just totally love the universal probe libray from Roche. You get 100 probes covering the entire genome and just order primers fitting the probe (extremely easy software on the website). It is a one time investment but then you can PCR every gene you can think of. I compared the system to traditional taqman primer/probes and it works amazingly well! I would recommend it to everyone (and I am not being paid by Roche to say this (unfortunately..))