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qPCR - qPCR problems.. (Dec/14/2009 )

HI...
actually I need some advices from the forum members...
i plan to do the qPCR for several gene of interest (GOI)...
and i plan to do the relative quantification...
from my reading, i have to test the primers efficiency before i start the real assay..
to do the primers efficiency, i have to generate the standard curve for each pair of primers..
from my reading, i found out that normally researcher will use the 10-fold serial dilution..

so, my questions is :
1) can i use 2-fold @ 5-fold serial dilution of my template??? because i have tried 10-fold dilution but the results is not so good..i think because of my GOI is not highly expressed because the Cq value for 100ng (highest concentation) is ~23..

2) besides that, do i need to run the assay (in the same running process) for the reference gene each time i run the GOI??? (i mean, if i have 5 GOI, so, i need to run it 5 reference gene???)..i asked this because i have several GOI and several samples...so, i cannot fit all the GOI and reference gene in the same running process...

really appreciate if someone can help me...
thanks..

-fibonacci-

Are the primers made-to-order or inventoried? If inventoried you often don't always have to test primer efficiency as this is guaranteed by the manufacturer

-Baars01-

actually all the primers i designed by using software and then i order with the manufacturer in my country...
so, that's why i have tested all the primers..

-fibonacci-

1. Yes you can use other dilutions, no problem. I always use 5-times dilutions, and used 2-times dilutions before. Your gene is a low-expression gene.

2. No. You can run the each GOI and each reference separately (in different runs)

-chrisbelle-

fibonacci on Dec 15 2009, 02:48 AM said:

2) besides that, do i need to run the assay (in the same running process) for the reference gene each time i run the GOI??? (i mean, if i have 5 GOI, so, i need to run it 5 reference gene???)..i asked this because i have several GOI and several samples...so, i cannot fit all the GOI and reference gene in the same running process...

really appreciate if someone can help me...
thanks..


I always include a single genomic DNA sample (same one I used for the original primer testing standard curve) in all my QPCR reactions. This will make sure that everything is going as it should (you should always get the same value for those wells). You can run your references and GOI separately if you don't have enough room on one plate, though I like to include at least the described control reaction with all the primers on every plate if I'm going to try to do relative quantitation across plates.

In general, I would prefer to run all the primers on a given sample on the same run. E.g. if you have 10 samples and you want to measure GOI and control on all 10, but you can only fit 10 wells per reaction, do the first 5 samples with each of the two primer sets on run 1, then do samples 6-10 with each of the two primer sets on run 2.

-jojoziggy-

jojoziggy on Jan 4 2010, 06:09 PM said:

In general, I would prefer to run all the primers on a given sample on the same run. E.g. if you have 10 samples and you want to measure GOI and control on all 10, but you can only fit 10 wells per reaction, do the first 5 samples with each of the two primer sets on run 1, then do samples 6-10 with each of the two primer sets on run 2.



I would recommend to do it the other way round. always try to pack all samples on one plate if this is possible. You are mostly interested in comparing the expression of your GOI between your samples therefore it is easier if all your samples are on one plate. If you spread your samples on more than one plate you also have to adjust for inter run variation, that means you have to include an inter-run-calibrator (IRC) on every plate that doesn't make the calculation simplier and increases the overall error.


This is explained in detail in this publication: http://www.ncbi.nlm.nih.gov/pmc/articles/P...02/?tool=pubmed
especially in figure 2.

-tea-test-