The objectives of this study is to check what gene is suitable for creation of a standard,to establish real time PCR as a high throughput technique for copy number work and to optimize / prepare a good standard for absolute quantification analysis for this work.
We designed two strategies for the optimization of qRT-PCR. The first one is through Southern blot copy number correlation with qRT-PCR. The second is through the creation of standard using a plasmid carrying the construct that was used in the plant.
In lieu of the first objective to find a gene suitable for doing copy number work we encountered problem with NOS gene.
We encountered problem like this using plasmid standard. Please look at the illustration.
One proposal to solve problem is the use of MS2 RNA. We still haven't tried it yet.
We did the serial dilution of the samples following the computation by Applied Biosystems. Click the link for computation.