Ct Values of NTCs (SYBR Green)

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mithu_du_bmb
mithu_du_bmb's picture
Ct Values of NTCs (SYBR Green)

We are measuring the expression of some genes in humans using RT-PCR (SYBR Green). In the past, the Ct values for NTCs were negligible, but now we are getting some values and some time as strong as the gene. We changed all the reagents, including primers, fearing contamination, but results are the same.  Can anyone help us?

Ivan Delgado
Ivan Delgado's picture
 

 
Hi mithu_du_bmb,
Did you check to see if the block of your real time PCR instrument is not contaminated? There should be a way to do this using the software of your instrument. If you find background fluorescence on your block, typically all you need to do is clean it up using Q-tips and 70% ethanol.
Alternatively it is always possible that you did not get rid of the contamination, even if you changed all your reagents. One way to test for this is to have someone else that has a qPCR instrument to run your experiment. If they get the same background (in their clean instrument) then it is your reagents. If they do not get any background then it may be your instrument, or your technique. 
Good luck
 

bejoy
bejoy's picture
hi,

hi,
contamination could be the key issue here,check on your instruments like centrifuge, work station and decontaminate them .Then you can swab all the work areas and run pcr to see if you can get cts, if so then you re-decontaminate.You can also check on how you load your controls if they spill over then you are likely going to get a contamination.if the primers were prepared at the same time there could be a blanket contamination of all the primers so you might be forced to prepare new primers for your PCR.

Ana Rita
Ana Rita's picture
Hey,

Hey,

If clean up everything does not improve your results, just try to decrease the concentration of your primers! Is not surprinsingly that you have a signal for your NTC. How different are the Cts from your NTC and your lowest concentrated sample? Check a recent publication on guidelines for qPCR (The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. by Bustin).
Good luck!