What does a CT value mean in PCR?
The Cycle Threshold (CT) represents the number of amplification cycles required for the fluorescent signal to cross the detection threshold, indicating the presence of a detectable DNA or RNA target.
Why is a low CT value important?
A low CT value indicates a high amount of nucleic acid template in the sample. It reflects strong amplification efficiency and is often associated with high target gene expression or high viral load.
How can I improve CT value accuracy?
Ensure sample purity, use validated primers, maintain accurate pipetting, and avoid PCR inhibitors. Calibrating your qPCR instrument and following standard curves can also improve CT precision.
Is CT the same as Cq or Cp?
Yes. CT (Cycle Threshold), Cq (Quantification Cycle), and Cp (Crossing Point) are equivalent terms used in different qPCR analysis software to describe the same quantification parameter.
What is a good CT value range in qPCR?
Generally, CT values between 15 and 35 are considered reliable. CT < 20 indicates a high concentration of the target gene, while CT > 35 may reflect low template quantity or background noise.
What factors affect CT values in real-time PCR?
CT results can vary due to template quality, primer design, reaction efficiency, reagent composition, thermal cycler calibration, and fluorescent dye performance.
How do CT values relate to viral or gene quantification?
CT values are inversely proportional to the amount of target nucleic acid. Lower CT = higher concentration of the gene or virus; higher CT = lower quantity.
What is the difference between absolute and relative quantification in qPCR?
Absolute quantification uses a standard curve to determine the exact copy number of a target gene, while relative quantification compares CT values of a target gene to a reference gene (housekeeping gene).
What are reference genes in qPCR and why are they important?
Reference genes (such as GAPDH, ACTB, or 18S rRNA) are used to normalize gene expression and correct for variation in RNA quantity or reaction efficiency, ensuring accurate CT comparisons.
What does a high CT value (>37) indicate?
A high CT value suggests low target quantity, poor amplification, or possible inhibitor presence. It can also indicate background fluorescence if the signal appears too late in the amplification cycle.
How do you calculate gene expression from CT values?
Use the ΔΔCT (Delta-Delta CT) method:
ΔCT = CT(target) – CT(reference), then ΔΔCT = ΔCT(sample) – ΔCT(control).
The fold change = 2^-ΔΔCT, which provides relative expression levels.
What is the ideal amplification efficiency in qPCR?
Ideal PCR efficiency ranges between 90–110%, corresponding to a slope of -3.1 to -3.6 in a standard curve. This ensures accurate CT interpretation and reproducible quantification.
What’s the difference between RT-qPCR and qPCR?
qPCR quantifies DNA directly, while RT-qPCR (Reverse Transcription qPCR) includes an additional step to convert RNA into complementary DNA (cDNA) before amplification. RT-qPCR is used for mRNA expression studies.
Can CT values be compared between different qPCR machines or reagents?
Not directly. CT values can vary depending on instrument sensitivity, fluorescence detection settings, and chemistry type. Always compare samples under identical conditions using the same master mix and machine.
How do melting curves relate to CT values in qPCR?
A melting curve analysis confirms the specificity of the amplified product. Sharp, single peaks indicate specific amplification, while multiple peaks may suggest primer dimers or non-specific binding that affect CT reliability.