Will there be late amplification (ct value) for negative control in qPCR?
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6 months ago
shail.nair05 ▴ 20

Note-This is a cross-post from https://www.researchgate.net/post/Will_there_be_late_amplification_ct_value_for_negative_control_in_qPCR

(Did not got any answers)

I want to know if my gene of interest has been amplified and has an acceptable level of expression (for the metabolic reaction). I have converted my RNA (no gDNA contamination) to cDNA using GSP and ran through ROCHE light cycler 480 for quantitative amplification (sample in triplicate with triplicate negative control (extracted RNA >>> ran cDNA conversion step without RT enzyme >>> added qPCR chemicals). The PCR was set for 34 cycles. Here is my ct value- Sample ct ct values

The positive sample ct values look good. But why does my control have late amplification (ct 21)? Is it due to Sybr green or machine noise? or does my control have some problem? If the ct values are normal, does just the ct values are ok to prove my aim (validation that the particular gene has been expressed), or do I need to do further calculations (I am new to qPCR)?

RNA QPCR cDNA • 466 views
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