defining ratio of alternative spliced gene with qPCR - (Sep/05/2013 )
Anpu on Mon Sep 9 08:37:32 2013 said:
doxorubicin on Fri Sep 6 08:56:04 2013 said:
A better approach would be to generate a standard curve for each PCR product and determine absolute quantities (ng) of each product.
doxorubicin: I am thinking about absolute quantification and I'm thinking about the standard curve.
What is the best way to make standard curve? I was thinking about synthesizing DNA of my gene and tha go from there
thank you for the help.
Sorry, I just saw this question directed to me. Assuming you're using a SYBR Green protocol...To make the standard curve do the following: Run the qPCR exactly as you plan to do for the final experiment (same RT from the same cells with the same primers and the same master mix). When the reaction is finished, upon the PCR plate, collect the completed reaction, and purify the product with a kit (Qiaquick PCR purification or equivalent). Use a nanodrop to determine the exact concentration of your purified product. Then prepare a dilution series 1:10, 1:100: 1:1000, 1:10,000, 1:100,000, 1:1,000,000 of the purified product in TE buffer. People usually add some sheared salmon sperm DNA for stability. Then test the dilution series using your qPCR protocol and see if it is linear with an efficiency <100%.
It is very likely that you will contaminate your pipetteman with purified PCR product. It might be better to use one from another lab to set up the standard curve.
Thank you for the reply. I ordered gBlocks from IDT. Just the other day the sails rep was here and we ere talking about it and he told me that this can be done with gBlocks. More and more people are using them and it great! They are not expensive, so my boss agreed :)
Regards and again tank you for the help!
I have another question about the absolute quantification.
Now that i have analyzed some samples i have noticed that some quantity numbers are surprising.
I have done one quantification with SYBR green (one plate) and another with TaqMan probes (other plate) no other way.
Do I have to change the treshold to the same value at bot runs or there is no problem if they are different? For TaqMan is lover that SYBR green!
Thanks for the help.
i stumbled upon another problem. With the absolute quantification where CTs in triplicates have just 0,1 CT standard deviation but when it is calculated to absolute molecule quantity the standard deviation with that can be greater than 10% between triplicates.
Does anyone have any Idea why? Ok i know the pipetting is probably the main problem but how do I explain that with statistics and in article.
Thanks for answers.