Hi all, I have been testing housekeeping gene stability in human dermal fibroblasts, ipsc and ipsc-derived cells for circadian studies. Using the same set of primers, the Ct for 18S rRNA is about 20, 8, 8 in the samples above.
In case you are wondering about whether it's the load. The assumed inputs were all 3ng cDNA/well in all qPCR set-ups. And the raw Ct levels for my other reference gene candidates from mRNA were quite similar, including bACTIN, GAPDH, HRPT1, RPLP0, TBP and YWHAZ. The samples were collected and proceeded separately across a few months, and run on difference qPCR plates due to space.
I used ThermoFisher Superscript IV with random hexamers for RT, and Thermo Fisher SYBR Green on a StepOnePlus Real-Time PCR system. The only difference in procedure I could think of is that I didn't put as much RNase out in the fibroblast samples during RT. Could there be a bias of transcription efficiency (or degradation) against rRNA, I wonder?
The cycle threshold (Ct) value in quantitative real-time polymerase chain reaction (qPCR) measures the cycle number at which the fluorescence signal of the amplified product crosses a predetermined threshold. The Ct value is inversely proportional to the amount of target nucleic acid in the sample. Lower Ct values indicate higher initial concentrations of the target.
For 18S rRNA, which is a commonly used reference gene, the expected Ct values can vary depending on the experimental conditions, the stage of the cell cycle, and the specific cell type. However, in general, for well-behaved and abundant housekeeping genes like 18S rRNA, Ct values often fall within a relatively narrow range.
For human dermal fibroblasts, the Ct value for 18S rRNA is commonly observed to be in the range of 13 to 20 cycles. However, it's crucial to empirically determine the Ct values for your specific experimental conditions, as they can be influenced by factors such as RNA quality, primer efficiency, and the specific qPCR instrument used.
Here are some general considerations:
Always refer to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines for best practices in qPCR experiments. Additionally, consult the literature and resources specific to your cell type and experimental setup for any reported Ct values for 18S rRNA in human dermal fibroblasts.
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