What are the possible reasons for not getting a Ct/Cq value for the amplification of some genes in qPCR?

Good Morning

One reason may be the copy number of the gene in your target cells.

Have you tried absolute quantification.

You can check the efficiency of primers for rtpcr by performing absolute quantification on serially diluted cDNA sample.

That may hint on required concentration as well the appropriate Ct values

Samiullah Khan

There could be several reasons why you are not getting a Ct/Cq value for the amplification of some genes in qPCR, including:

Poor quality of RNA: If the RNA used for cDNA synthesis is of low quality, it can affect the efficiency of the reverse transcription step and lead to reduced amplification of some genes.

Low abundance of the target gene: If the gene of interest is present at low levels in the sample, it may not be detectable by qPCR. This can be especially problematic if the housekeeping genes used for normalization are highly expressed.

Suboptimal primer design: If the primers used for qPCR are not specific or efficient, they may not amplify the target gene well, leading to a lack of detectable signal.

Inhibitors or contaminants: Some compounds present in the sample can inhibit qPCR amplification or interfere with the fluorescence signal. This can be a problem especially in complex samples like tissue or blood.

To troubleshoot the issue, you could try the following:

Verify the RNA quality by checking its purity, integrity and concentration. If the quality is low, you can repeat the RNA extraction and cDNA synthesis steps.

Increase the amount of cDNA used in the qPCR reaction to improve the sensitivity.

Optimize the primer design by checking for specificity and efficiency using primer design tools or by testing different annealing temperatures.

Dilute the sample to reduce any inhibitors or contaminants that may be affecting the qPCR reaction.

As for troubleshooting getting higher Cq values for housekeeping genes used in qPCR, you can try optimizing the annealing temperature, optimizing the primer concentration, or using a different housekeeping gene as a reference. Additionally, it's important to make sure that the housekeeping gene is not affected by the experimental conditions or treatments being studied.

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