Hi ! If you need to quantify a particular gene in a DNA sample with different genes, the best method that you can use will be by quantitative PCR (qPCR) by using specific primers of the gene. If your cDNA sample is pure with DNA from only one species, you can use absorbance or fluorescence methods (like PicoGreen) and quantify the DNA concentration of the whole genome and then calculate the number of copies.
You definitely need to perform quantitative PCR (qPCR) with specific primers of your gene of interest
.
Hi,
People usually work on gene expression with cDNA (Relative quantification, gene of interest vs house keeping gene)
Even if I don't clearly see the point to do this, you could also get the copies number for a target in cDNA by real time PCR (Absolute quantification, standard curve), the value will then be expressed in copies number / sample in your reaction (cop nb/ vol of your sample or cop nb/ sample's quantity if you can access its concentration).
(Ps: make sure your RNA samples were DNAse treated)
Hope it helps
1) A TaqMan or SYBR/Eva-Green based quantitative real-time PCR assay.
https://www.thermofisher.com/de/en/home/life-science/pcr/real-time-pcr/qpcr-education/taqman-assays-vs-sybr-green-dye-for-qpcr.html
2) DNA binding dye like Pico Green.
Download the QuantiTect® Whole Transcriptome Handbook - Qiagen
https://www.qiagen.com/no/resources/resourcedetail?id=6910f167-e4ae-43a2-b87d-26a74c55a75c&lang=en)
and follow instructions in Appendix C.
in cDNA sample you'll have an idea on the transcription of the gene, not on copies.
fred
Hi!
You can estimate the number of cDNA copies in your sample using qPCR or digital droplet PCR.
Einari
I agree with all the posts that tell you, that expression differences will blast every copy number determintion. Just a an example: think of two genes, both are single copy ... one of it has a 4 times more effective promoter ... this one will produce four times more transcripts. You see the prob?
If it is genomic, you should think about southern blot, which was log time the standard method. Today you can perform this without radionuclids. qPCR is another possibility as already mentioned. However, depending on your question, it might be more interesting, where the specific integration site is rather than the number of interation sites. Please give a little more details next time.
Thank you all for sharing your ideas, they will indeed be very useful :)
Actually I think that both types of information would be very useful for my future work, quantification of gene copie number and transcripts, so maybe a qPCR with cDNA as template to have an ideia about the effectiveness of transcription and another one with genomic DNA to quantify the number of gene copies.
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