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Science in Lab | Troubleshoot High Ct Values: qPCR Optimization in Three Steps

Are you having trouble with high Ct values and poor repeatability?

Find out whether issues are caused by low gene expression, and check out tips on how to optimize your experiment to resolve these issues!


What is a low-expression gene?

Low-expression gene usually refer to those genes with low reads per kilobase of exon per million mapped reads (RPKM) in a given sample. In qPCR assays, the Ct values of low-expression genes are often too high, sometimes accompanied by poor reproducibility between duplicate wells.. With high Ct values, reference genes in the system exhibit normal Ct values, while target genes have Ct values above 30 (dye-based qPCR).



Figure 1. Illustration of Low Expression Genes


How can we optimize qPCR assays for better detection of low-expression genes?

01 qPCR program


Use a three-step instead of two-step program or increase extension time to improve amplification efficiency.









Two-step program-Extended extension time-Three-step program-Extended extension time

02 Experimental procedure

A. Increase the number of replicate wells and remove outliers;

B. Add carrier DNA or RNA that neither reacts with the target gene nor interferes with the assay to minimize loss of target genes due to adhesion to pipette tips or tube walls during liquid handling.

03 Reaction system

A. Template: Poor template purity, inhibitors included, template degradation and low  input will lead to high CT values.


ü  RNA: Evaluate RNA integrity with agarose gel electrophoresis and purity with NanoDrop;

ü  Reverse transcription: Increase RNA template input amount;

ü  cDNA: Increase cDNA input or use a lower cDNA dilution factor (in theory, each 10-fold dilution increases Ct value by 3.3). When using a cDNA stock solution for qPCR, the input amount should not exceed 1/10 of the reaction system.


B. Primer: Non-specific amplification consumes primers and Taq DNA polymerase, reducing the chance of primers binding to target genes and compromising amplification efficiency.

Recommendation: Follow primer design principles to prevent non-specific amplification. Primer efficiencies should be between 90% – 110%.

Primer efficiency calculation

The target gene is diluted in different gradients (at least three dilutions) and used as a template for qPCR. Obtain autofit standard curves from the qPCR system, or import Ct values into Excel and plot the standard curves manually. Plug the slope of the standard curve into the primer efficiency formula:


C. Kits: The formulation of Taq DNA polymerase, buffers, Mg2+, and fluorescent dyes varies across different qPCR kits, affecting specificity, sensitivity, and amplification efficiency.

Recommendation: Use kits specially designed for detection of low-expression genes. Vazyme’s Taq Pro Universal SYBR qPCR Master Mix (Vazyme #Q712) is ideal for your low-expression gene testing needs.


[Vazyme #Q712: Robust Detection of Low-Expression Systems] Performance Showcase

More sensitive detection of low-copy templates

Assays on six 10-fold serial dilutions of a mycoplasma plasmid template demonstrate that Vazyme #Q712 displays excellent amplification curves and linearity for low-copy plasmids, and is capable of detecting as low as 1.75 copies of plasmids (0.35 copies /μl dilution, 5 μl input). Compared with an earlier version of our dye-based qPCR kit, Vazyme #Q712 has a lower Ct value at the limit of detection (LOD) of the sixth dilution (lowest concentration), indicating higher sensitivity.


Figure 2. Amplification Curves at Different Copy Numbers (Vazyme #Q712 vs. Vazyme’s Previous Kit)

Ct Value

Copy Number (copies/μl)

Vazyme’s Previous Kit


3.5 × 104



3.5 × 103



3.5 × 102



3.5 × 10









Amplification efficiency



Figure 3. Ct Values at Different Copy Numbers (Vazyme #Q712 vs. Vazyme’s Previous Kit)


More robust detection of low-copy templates

The detection rate of single-copy target fragments should follow Poisson statistics. In theory, 70% of 1.75-copy templates are expected to test positive, and 30% are expected to test negative. Low-copy (1.75 copies) mycoplasma plasmid templates were assayed using Vazyme #Q712 and Supplier A’s comparable product. Results show that both kits yield detection rates predicted by the Poisson distribution. Vazyme #Q712 has a detection rate of 75%, better than Supplier A’s product.


Figure 4. Detection of 1.75-copy Plasmids (Vazyme #Q712 vs. Supplier A)


Ordering Information

Easy testing of low-expression systems with superb amplification curves!

Product Name

Cat. No.


Taq Pro Universal SYBR qPCR Master Mix


500 rxns (20 μl/rxn)



Recommended Consumables

Use together with Vazyme’s consumables to obtain more reliable data.


Product Name

Cat. No.


8-Tube PCR Strips

0.1 ml 8-Tube PCR Strips (with Caps)


125 Strips

0.2 ml 8-Tube PCR Strips (with Caps)


125 Strips

96-Well Plate

0.1 ml Semi-Skirted 96-Well PCR Plates



0.2 ml Semi-Skirted 96-Well PCR Plates



Sealing Flims

Pressure-Activated Plate Sealing Films



Self-Adhesive Plate Sealing Films





Learn more information or place order online, please visit: www.vazyme.com