QPCR, where to start? - optimizing primer and template (May/17/2006 )
Hi everyone,
I am new to QPCR and I was wondering what I would need to do first in preparation for it. I designed and ordered my primers but have not yet figured out where to extract my cDNA from. I am looking at 3 genes that associated and are involved in focal adhesions and want to look into their gene expression level. I read from one paper that talked about the association of two of these genes and used DNA extracted from rat brain. They also talked about studying these genes in pancreatic and bladder cancer cell lines. Anyone have any idea where I would extract the DNA from?
Also, once I get my template cDNA what should I do before starting the actual QPCR? I read that you need to optimize the primer and template concentrations but I'm not exactly sure what that means. I know we need to run dilutions of the primer and templates and melting curves but how do you do that exactly? I also read that 2uM of primer is a good amount to use...is that too much? The product info that comes with my primers that I ordered only says there is only about 40nmol in my primer...or am I reading it wrong? Does that mean there's 40nm in one ul or in the whole tube? Sorry but I've never dealt with this before. Also how do we figure out how many ug/ul we have in our primers? Does the product sheet tell you or do we figure that out? I usuallu use around 100ng of template for normal PCR reactions, so do I test the template concentration around that range or is it different for QPCR?
1. Since you are runing QPCR for gee expression, you should be extracting RNA rather than cDNA. With the extracted RNA, you can run either one-step or two-steps RT-PCR.
2. Since you are doing relative quantification, you should amplifying a hounskeeping gene as internal control.
3. Testing your primer...... first strat with single pair primer set, run at basic condition: 1x buffer (10mM KCL), 1.5mM MgCl2, 250uM dNTP, 0.5uM of each primer, 1.25 unit of Taq..... (SYBR Green?)
Run on gel as well to confirm your PCR product.
You can optimize you assay by changing primer concentration and MgCl2 concentration.
4.In you primer information sheet, there should be a column writen with " add x.xx ul to become 100uM". Reconstitute you primer accroding to that volumn. make aliquote with working concentraton of 10uM.
5. Your template should not be more than 1000ng.
2. Since you are doing relative quantification, you should amplifying a hounskeeping gene as internal control.
3. Testing your primer...... first strat with single pair primer set, run at basic condition: 1x buffer (10mM KCL), 1.5mM MgCl2, 250uM dNTP, 0.5uM of each primer, 1.25 unit of Taq..... (SYBR Green?)
Run on gel as well to confirm your PCR product.
You can optimize you assay by changing primer concentration and MgCl2 concentration.
4.In you primer information sheet, there should be a column writen with " add x.xx ul to become 100uM". Reconstitute you primer accroding to that volumn. make aliquote with working concentraton of 10uM.
5. Your template should not be more than 1000ng.
I don't see that column on the sheet, all I see are columns denoting the nmol, ug, mw. etc. I understand we need a housekeeping gene as a control, but what is a calibrator then? Where do we use these? And melting curves and CT curves, how do we run those? I am really overwhelmed with all this information, I feel like I am getting bits and pieces from everywhere but never the whole picture or enough to know what to do.
http://www.protocol-online.org/forums/inde...showtopic=15704
work through the posts on this forum, sometimes there are some helpful links
another thing, it's pretty impossible to go through the whole thing in a format like this, it's a very big topic and it takes quite a bit of work to get it rolling
please see some of the links I pasted into that thread
work through the posts on this forum, sometimes there are some helpful links
another thing, it's pretty impossible to go through the whole thing in a format like this, it's a very big topic and it takes quite a bit of work to get it rolling
please see some of the links I pasted into that thread
Thanks, I have read through the posts before. I realize it would be cumbersome to explain the whole process, as it is cumbersome to read about it, but I read a bit more about it and figured out how to reconsitute my primer and make a 10uM working solution. Now I am trying to test out my primers to check for PCR product then after maybe try to optimize it with different template and primer concentrations? I am just using 100ng template and 10uM primer in a 100ul reaction for now, I see a lot of different opinions on what template/primer concentrations to use however. I hope I am heading in the right direction.
it's all about User Bulletin #2
that is a big pain to read and there is some info you may not need, but the sections on optimizing, setting up a standard curve, and validating ddCt are golden