new to real time PCR- where to start? - help me (Apr/20/2006 )
Iam planning to use real time PCR to estimate the levels of certain mRNA in my bacterial isolate.
Iam new to real time PCR and just started reading about it. There seems to be many ways to do it. Iam confused about choosing the right method for my work. should i go for taqman method or syB green based detection?
the other problems i have are....
I have to go to a different city to use the rtPCR machine. I heard that trasporting RNA is tough as it is very unstable. so will it be safe to convert the RNA to cDNA and transport it?
how long and at what conditions can RNA and cDNa be stored?
Please suggest me some literature to read more about real time PCR experimental protocol. I will be using Rotorgene 3000 for my work.
i think thats it for now.thanks
some resources for you:
this one...very technical if you're a raw beginner, but once you've got the basic concepts down this guide can be your friend when you are trying to get started with runs
that one (go to bottom of page, look at all the stuff in "further information")
this one - try some of these online tutorials (you can ignore the product plugs and get some good info)
this one you may also find to be helpful
and yeah, your best bet is to convert your RNA to cDNA rather quickly; cDNA is much more stable (I keep it in nuclease-free TE, in the fridge for up to a month or the freezer for many months..some people store it in the freezer for years). then if you want to transport your samples, you can just put them in a cooler. you still don't want to freeze/thaw them very many times, but they will last a good while if you have have nice clean technique
another thing you might want to do...get a sales rep from your local office, from Qiagen, ABI, Stratagene, Invitrogen, BioRad, Roche...any big name that sells qPCR stuff, and have have them come give you a tutorial
one last consideration...I don't know how much you've worked with RNA? www. ambion.com has a lot of really good information about working with RNA (their website is a bit cumbersome but it's worth it); they also have some information on how/why to choose a housekeeper for your system...some great stuff that can help you
also, look through posts here. this comes up quite often and there are alot of good tips and advice discussed here
thanks a lot.thats very useful.
yeah. i dint work with RNA much. just did few RNA isolations from e.coli five years back.
i read through the material you suggested. thanks. it was very useful. But, i need some clarification.
i have decided to go for sybr green based detection. all the companies say, they have optimized buffers for RT step and real time step. Iam interested in estimating the levels of overexpressed mRNa and mRNA of a particular gene in chromosomal DNA of bacteria. Will the optimized buffers work well for such applications or should i go about optimizing each conditions in buffer?
what should be the primer length i should use? can i use the PCR primers used for amplfying the gene and has overhangs in the 5'end. My primers are around 20bp with 10bp complementation?
and finally, what should be the purity of the primers
I would not use your cloning primers; I would design primers just for the purpose of qPCR
I use standard desalted synthesis from Invitrogen or Operon
ABI has some good recommendations on primer design. It is good to have 18-23 bp each, Tm's about 60ish, even G/C to A/T ratio (I think; you will want to check their site to be sure I am quoting this correctly
An important thing to consider is that you want small amplicons...in the 50-150bp range...for this technology to work the best. I shoot for about 100 and try to keep all my amplicons pretty close to the same length
the buffers that come with your RT and SYBR green kits are optimized and you shouldn't have to mess with them much if at all. There is a slim chance that you may need to adjust the MgCl2, but I find in the long run it's easier to try another primer set
Also, the places where you will have to optimize are likely to be template amount added, and primer concentration. That 'user bulletin #2' link that I sent you can give you some good recommendations there. Please pay careful attention to the stuff on amplification efficiency.
I would also recommend that you sift through other threads in this subforum; there is a great deal of useful information that has been covered when others have started.
thank you very much for your suggestions.