Hi everyone,
I'm currently running RT-qPCR on my cells (with Sh-Scramble and Sh conditions). Previously, the Ct values for my Sh-Scramble cells were consistently in the range of 24-25.8 across different cell lines. However, in the last three qPCR runs, the Cts for these same Scramble lines have jumped to 28-29, which is quite puzzling.
Here are some things I've checked already:
- Housekeeping gene (Actin): Ct values are stable and consistent.
- RNA quality: RNA integrity is good (all ratios > 2.0).
- qPCR machine: No issues with the equipment.
- Melt curves: No changes in primer melt curves compared to previous runs.
Interestingly, the Ct values for the Sh condition have always been in the 27-28 range and haven’t changed. The problem is that this shift in my Scramble control is now causing unreliable results in the delta-delta Ct calculations, showing that the Sh cells might be expressing more than the Scramble control, which is obviously counterintuitive.
Any insights into what could be causing this would be greatly appreciated!
Thanks!
It's very strange that your Actin has stayed consistent but your target gene has changed Ct value under a negative control knockdown. Is it possible some Ct calling setting on your qPCR machine has been changed without your knowledge? On our machine there's an option to switch between High Sensitivity and High Confidence; I don't really understand how this setting changes Ct calling, but if I switch between these options then some of my Ct values change but not others.
Are you using cells at similar passage number across your assays? Are the cells cultured under shRNA KD condition for the same amount of time across your assays? You said the RNA quality is good, but are you confident you're using the same RNA quantity across the assays?
Which shScrambled are you using? Some negative control shRNAs have been shown to not actually be non-targeting, eg Article Non-targeting control for MISSION® shRNA library silences SN...
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