We have Corbett's RT PCR in our lab but I am having difficulty in analysis of data and I am getting amplification in negative control. Can anyone help me ?
Hi Brijesh
We have a qRT-PCR machine of Corbett Research company in our Institute..and it is working quite fine....I would recomment go through the manual of the company thoroughly before starting the experiments...and for getting amplification in negative sample...there has to be some role of your samples or methodology.than the machine itself..
bye
which kit you used ,did you use syber green dye or taqman probe or what kind of fluorescence you used give me some detail to help you
i think you use syber green ....may be this reason to getting amplification in negative control...
yes you are right..i had used sybr green..and @ ajay saini i had read manual and all the things but i dont know how to analyse data after run...Would you please help me to analyse data for corbett's software(rotor gene 6000)?
Hi Brijesh
What eaxctly is the problem you are facing.....Is the machine not displaying anything, or your are not able to access the output file, is it not showing the amplification and melting curves, are you not able to get Ct values,, or everything is fine and you are not able to analyze it using the software......kindly elaborate the exact nature of the problem you'are facing....mean while I have some suggestion..
1) Is this the first time somebody has used this machine in your lab/institute?.....If yes, I would suggest you to get in touch with the Company technical people and they can show it to you....
2) if some researcher has used it previously use his/her help....to analyze your data.
3) If you have gone thorugh the manual..then it should not be much of a problem using any qPCR software......Basically you have to label your samples as control and treated and the genes as internal control and gene of interest., and -ve controls....in addition, if you have a range of standard concentration they are also needed for absolute quantification....
.If you are able to get/retrieve Ct values ..try to analyze them in Excel or some other software and see if everything is OK.....you may be missing out something tricky in the software
let us know the nature of the problem and researchers on RG will give you way out..
bye
Thank you so much Ajay Saini for your concerned to my problem.Actually I am the first one in my institute who is using this instrument. So i want to learn everything from basic. The thing is that we have got the data but i dont know how to check how much my gene of interest is expressed compared to control?
I use to work with the Corbett rotor gene 6000 and quick tips to analyse your data in excel:
-First in the main window (log graph) verify that you have a good amplification, the s shape curve. Adjust the threshold to make sure you are using the data within the linear range.
-Second check your melt curve to make sure you have only one amplicon. You should have only one pick if the primers are good, binding only one location. If not you data is not reliable.
Then copy and paste your data in excel and then start analysis.
-check your standard curve and make sure it is ok (R-value)
-obtain the standard curve equation and derive the concentration of your gene of interest in each sample.
-Normalize your data using an internal control (housekeeping gene) in each sample.
And that is it. But like other suggested you have to follow the manual it will tell you exactly how to do it.
Good luck
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