I have an unknown Chikungunya Extracted RNA? How can I determine a single copy number of a target gene from the virus and drew a standard curve to test the Limits of detection of my assay?
I think with qRT-PCR. comparing RT-PCRs of dilutions of the amplified RNA-DNA fragment sequence which you known the molarity.
Firstly of all you need a standard Chikungunya RNA extract. You can buy an assay kit with standard positive control. Then the accurately quantified standard is serially diluted in increaments of 3-10 fold dilutions and the range must be sufficient to cover the quanties of viruses expected in your test samples. The standard curve is generated from average ct values of each dilution plotted against the absolute amount of the standard. The standard curve is then used to compare the ct values of experimental samples. this gives an estimate of the amount of target in experimental samples.
Limit of Detection
Limit of detection is the amount of in the highest dilution with a ct values. Because you will notice that on your standard curve there will be some ditutions without a CT value.
Thank you for the answers, Am trying to develop a positive control that can be use as standard curve for may assay. I only have the cultured sample of Chikungunya which I obtained with no information abut the virus number.
PCR amplify your gene of interest. Purify on an agarose gel, quantify at OD 260. Based on the concentration and molecular weight you can find out copy numbers and you can use it as standard.
Careful not contaminate your reagents with amplified product
That's Great, I was thinking of using the cloning methods to have a stock and used as a positive control.what you think?
Cloning the gene of interest is a permanent solution.
once a gene is cloned and being used in PCR, contamination problems (false positive) starts
Careful not contaminate your reagents with cloned product
But if I wanted to use my gene as a positive control cloning is my only solution.
Can you explain to me why false positive start please?!
The following links may be of use to understand
http://www.annclinlabsci.org/content/34/4/389.full
Good read for anybody working with PCR
http://bitesizebio.com/20773/clean-up-your-act-how-to-clean-up-pcr-contamination/
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