We have undertaken the genetic transformation of Eucalyptus. We want to find the copy number of the transgene in the different transgenic lines. Could anyone please explain this using Real-Time PCR. We do not have the facility for southern blotting.
Go throigh this paper
Cloning the broccoli HMG-I/Y gene as an endogenous
reference for transgene copy number determinations
using real-time PCR
Chun-Hung LIN, Jia-Yuan HUANG, and Long-Fang O. CHEN*
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 11565
To calculate the number of transgene copies and copy
number, they used the equation designed by Weng et al.
(2004) below:
[1] X0/R0 = 10[(CT,X – IX)/SX] – [(CT,R – IR)/SR]
I suggest to desing a very specific primers for transgene check the efficience by curve and melting point. And make some correlations between transgenic lines as an example put 30 cicles and check the curves and calculate the diferences of reached lines to measure an aprox. of copyes (be cautelous for some positive errors).
The best way to perform this kind of analysis is use a calibration curve for an absolute quantification. You need to use the qPCR primers (80-150 pb each) of your transgene and a serial dilution of the plasmid or construction of your gene. Then you just interpolate the Cq of the transgenic DNA and analysis the copy number per haploid genome. This way is useful when you analyse transgene copies in lines or organism with more than 2 genomes as wheat for example .
In other hand you can use a relative quantification method, but in this case you must find a gene with one copy in the genome of the organism. Then using a mathematical algorithm that integrate Cq (or Ct) and slopes and efficiencies you are able to predict the copy number of the genome... for example if the ratio of the final values between the transgene aand the unique copy gene is 1...then you can said that you have one copy.
Attached you will find two papers that explain the details
Specific assays are obviously crucial. However, you also need controls. The easiest case if you have negative and positive control transgene organisms with known copy number. You can use these as calibrators to determine the copy number in the organisms with unknown copy number. If you don't have these control samples, you will need to make your own 'samples'. We recommend to order synthetic templates (ss oligonucleotides, e.g. first and last 30 bases of your true amplicon) (importantly, you need to order these with another provider than the one you order your primers from) for your gene of interest (transgene) and endogenous reference gene and mix them in 1:0, 0:1, 1:1, and possibly also other ratios, which can then serve as your control samples with known ratio of the copies of the transgene and reference gene.
We have a Methods paper on qPCR based copy number determination that may be useful: http://www.biogazelle.com/resources/articles (D'haene et al., 2010, Accurate and objective copy number profiling using real-time quantitative PCR, Methods. 2010 Apr;50(4):262-70).
Did you even consider in using digital PCR for copy number determination? At Biogazelle, we routinely use ddPCR for copy number determination, also in transgenic organisms.
Or you could explore the following equation in the attachment here. It worked beautifully for a transgene research group in Cuba recently. The key (once again) is to not assume that the exact same amplification efficiency for plasmid (or other) standards holds true for the same target assessed in the biological sample isolate context. This is an important technicality not often mentioned - and, I think, swept under the rug all too often.
It may have to do with cycles 1 thru 5 of every PCR/qPCR/RT-qPCR (and the initial context of the target/template introduced); the very region of every PCR/qPCR/RT-qPCR within which the process is just getting up [kinetically] on its feet for the very first time before the desired blunt-end amplicons are being generated in a vast majority by the intended PCR/qPCR/RT-qPCR...
See the publication:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2891329/
Go throigh this paper
Cloning the broccoli HMG-I/Y gene as an endogenous
reference for transgene copy number determinations
using real-time PCR
Chun-Hung LIN, Jia-Yuan HUANG, and Long-Fang O. CHEN*
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 11565
To calculate the number of transgene copies and copy
number, they used the equation designed by Weng et al.
(2004) below:
[1] X0/R0 = 10[(CT,X – IX)/SX] – [(CT,R – IR)/SR]
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