SYBR green PCR - (Jan/30/2008 )

Hi,

Ok so slowly slowly I'm getting ready to start my qRT-PCr I'm going to use SYBR as Taqman is too expensive. I'm the only one doing it so I have lots of questions..(previous posts) but any help with my newest ones would be great!

Any tips for choosing a house keeping gene? I keep reading contradictory info on what genes are good to use and which arent. ALso how do you all choose what company you buy the SYBR mix from, PCR plates, covers to use etc... are they all much the same?

Thanks!

concerning choosing housekeeping gene. for this we would need more information about what you want to study. are you working with cell lines or tissues?
if you are working with cell lines there is maybe a good chance that someone has already evaluated a suitable HKG in that specific cell line, just do same literature research. also your experimental approach is important. are you comparing different cell lines? eg. malignant vs. non-malignant or are you treating one cell line with a drug and compare treated vs. non treated? for the latter, common HKGs like GAPDH or HPRT should do the job, just make sure they are not affected by your experimental manipulations.

Tissues: this needs some more evaluation, especially if your are studying tissues from different individuals or different stages of development,.... it is good practice to normalize against 2-4 different HKGs, preferable from different physiologic pathways to make sure they are not co-regulated. there are also commercial kits like genorm available or you can work with your own primers and evaluate with an applet like genorm or normfinder.
i think implementing such HKG evaluation in your study is a big quality improvement of your data.


concerning reagents and plastic ware: again, some additional information would be useful. which instruments are you using? For ABI machines i can recommend the ABI plates (#N801-0560), they work really well and i have tested plates from other suppliers with very bad results. however, take care that you need to order optical plates and caps (or foils).

i don't have experience with sybr reagents because i'm doing taqman. if you have the time, get some free samples from the relevant manufacturers and compare how they function on your instrument. if all are comparable, take the least expensive. if not take the best and negotiate with the sales agent. usually, every company will give you some discount.

Hi,

thanks very much for that!!

I'm working on rat neural stem cells (from tissue then using the primary cells) looking at gene expression and then treating the cells (at the start) and checking the expression (at the same time points loking for a change), so the fact that I should use more than 1 HKG is very informative thank you!

I will be using a BIO-RAD machine and the others I have spoken to all use an ABI so use the ABI stuff. I have looked at te BIO_RAD site and they seem to sell plates etc, they have claer ones and 'white'. they say this will effect the rsults due to floursence from other wells.... I just dont know anyone who has used them before or know if they're the best to use.... I will ask for some samples!

THanks for your help.

Does any one have the experience of using white pcr tubes Vs clear ones? Presumably the white ones are more expensive - but are they worth it? Is the signal really tha much stronger??

i think the white ones give better reflection and therefore a stronger signal. i don't have a direct comparison if they are really worth the price but they have one disadvantage: you don't see how much volume you have pipetted in the well, i control often when working with the transparent ones in case i have forgot some well.