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Genomic DNA copy number qPCR - Help with control (May/05/2009 )

I want to check the copy number of a gene I'm working on in a panel of human cancers to check for LOH and/or amplification at the genomic DNA level. However, I don't have any paired normal DNA for comparison, which is usually used in these types of studies studies. I've come up with 2 potential alternatives and would like your thoughts and comments and if you have any, alternative suggestions.

1) Relative quantification using RNase P or Gapdh as housekeeping. I have some WT human DNA to use, but the problem is that I will have no idea if the RNase P locus also changes in copy number and could make the analysis complicated/mask effects. Most of my tumors are of high grade and could be expected to have significant genomic instability.

2) Use a standard curve approach, without an endogenous RNase P etc control. I'll make serial dilutions of my WT DNA, and load equal amounts of DNA from my tumor samples (we have a nanodrop in the lab, although maybe picoGreen would be better for this type of application). Then I can directly compare the sample Ct to that of the standard curve and calculate copy number.

Suggestions?

-miRNA man-

hi there!

I think approach number 1) is a good idea, but you would of course need to be sure that your housekeeping gene(s) are stably expressed in your sample AND controls.Therefore it would be adequate to get primers for several genes, make a testrun and pic the most stable ones.

very helpful would be this paper:
"accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes" by vandesompele et al.
it has a table were it lists several good housekeeping genes and for which tissues they are most stable.
primer-sequences can be obtained from this paper or, in general ,from this website:
http://medgen.ugent.be/rtprimerdb/

I m not sore sure with option 2).
it is for sure a good idea to make a dilution row of the cDNA, for instance to evaluate primer efficiency and overall PCR-reaction efficiency and stability.
also it is a good idea to normalize the cDNA input....but this not so easy: how do you measure the cDNA concentration?nanodrop is not enough, because it also measures the leftover Dntps and the enzymes. so you would have to purify the cDNA before measuring......this again would probably lead to a loss of total cDNA.
Much more common and preciser is the method to subtract the "background" of a stablyy expressed housekeeping gene.

-asdfyrr-

Thanks asdfyrr. I'm going to do qPCR from genomic DNA, not RT-PCR from cDNA. Does this change your mind at all? I just wonder that there are so many chromosomal changes in cancer, how can I be sure that a gene that is usually 1-copy (i.e. RNase p) is maintained as such? Maybe I should just try a couple of different controls as you suggest.
Attached File

-miRNA man-

hi again ,

sry i missunderstood something there. of course it makes a difference if you analyze cDNA oder gDNA. im sorry i somehow thought you would look at the expression lvl to see if genes are overexpressd in your samples rather then checking for chromosomal copy numbers of your target genes.

solution number one has, like you said, the problem that you dont know if your reference gene has the same copy numbers in your cancer sample.

if you can precisely measure the dna content then your solution number 2 might be fine i guess. or you could use it to check if your reference gene has the same copy number in al samples and use it in another reaction for normalization.
but this might be unnecessary

-asdfyrr-

Thanks so much for your help. I did more literature searches today and found a paper and some references that say Line-1 retrotransposable element is consistent at the DNA level between normal and tumor samples of the cancer type I'm looking at, so I'm going to go with that with rel. quantification and see how it goes. Thanks again!

-miRNA man-

glad i could help. this qPCR stuff is kind of tricky. let me know how its wotks out and if you have anymore troubles.

yrr

-asdfyrr-

Hi, im dealing with the same topic. I have some DNA samples from patients`s lymphocytes that show genomic amplification of certain regions of a gene, as detected by MLPA (Multiple ligation probes assay). I want to confirmate these amplifications using qPCR. In my MLPA results i have a control DNA that shows a normal copy number of these regions. So here is the design i have in mind:

DNA Region 1: Shows genomic amplification in samples but normal copy number in control.
DNA Region 2: Shows normal copy number in samples and control.

Here are my questions:
Can I do the normalization of samples and control using the amplification of region 2 of the respectives DNAs and after calculate folds of amplification of region 1?
Does qPCR is able to detect an amplification of two times a locus???

thank you, waiting for your help.

-MLPA-

hi mlpa,

as is said above I am not really an expert when it comes to qPCR on a genomic level because i always used cDNA as a template that i got from mRNA extractions.

but in principle you should be alright with the samples and controls you have. if you are sure that your control region is normal in copy numbers than you can use it to normalize your experimental Data of your DAN region 1

-asdfyrr-

miRNA man on Tue May 5 19:14:00 2009 said:


I want to check the copy number of a gene I'm working on in a panel of human cancers to check for LOH and/or amplification at the genomic DNA level. However, I don't have any paired normal DNA for comparison, which is usually used in these types of studies studies. I've come up with 2 potential alternatives and would like your thoughts and comments and if you have any, alternative suggestions.

1) Relative quantification using RNase P or Gapdh as housekeeping. I have some WT human DNA to use, but the problem is that I will have no idea if the RNase P locus also changes in copy number and could make the analysis complicated/mask effects. Most of my tumors are of high grade and could be expected to have significant genomic instability.

2) Use a standard curve approach, without an endogenous RNase P etc control. I'll make serial dilutions of my WT DNA, and load equal amounts of DNA from my tumor samples (we have a nanodrop in the lab, although maybe picoGreen would be better for this type of application). Then I can directly compare the sample Ct to that of the standard curve and calculate copy number.

Suggestions?


Hi,

You could use the second approach and you can able to pick the sample which has two copy numbers among your sample and use it as calibrator.

Using LINE/SINE elements as ref should be a bit tricky because some of these elements are polymorphic and correlated with cancer.

CK

-Kalpana Venkat Krishnan-