during my experiment I got non specific amplication in NTC, does that affect the values of other samples and targets? do we need NTC everytime we run the plate?
Hello Parthvi Joshi
Yes, NTC is required. NTC wells are essential for detecting contamination or non-specific amplification, and a minimum of two NTC wells should be included per reaction plate.
Please note that signal detection in the NTC is unacceptable as this means the result in the experimental assay may be falsely inflated or even worse, a false positive. When a signal is detected in the NTC, it is often assumed that the problem may be laboratory handling error. If handling error has been ruled out, then often the only conclusion which can be drawn is that the reagents may be contaminated.
To avoid this problem, general sterile procedures should be followed, such as not opening vessels after reactions or doing so in a separate laboratory area with separate equipment and routine decontamination of laboratory space and equipment.
I would suggest you use Melt Curves to determine if the NTC amplification is caused by carryover contamination. If this is the case, then the products in the NTC reactions will be the same species as the intended target and the melt profiles will match accordingly.
If contamination is detected, then all reagents (which includes primers, water, qPCR mix) should be replaced, equipment should be decontaminated with a 10% bleach solution, and sterile procedures should be used for future experiments.
Best.
I would also recommend NTCs, I've seen NGS libraries get contaminated with PCR amplicons. The only reason we figured it out as quickly as we did was because of the NTC sample being contaminated.
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