Science in Lab | Improving the Accuracy of qPCR for Low-Expression Gene Detection

Researchers frequently encounter issues with data reliability due to high Ct?values and poor data repeatability in qPCR assays, particularly when detecting low-expression genes. These issues often stem from intrinsically low levels of gene expression, while the accurate detection of these low-expression genes is critical for biological research and clinical diagnosis. This article aims to help you improve the sensitivity and accuracy of qPCR for the detection of low-expression genes by optimizing assay conditions.?

What is a low-expression gene?

In general, a gene with low reads per kilobase million (RPKM) in a given sample is called a low-expression gene. In qPCR assays, low-expression genes yield high Ct?values, sometimes with poor repeatability between replicate wells. Specifically, in the same dye-based qPCR reaction system, reference genes exhibit normal Ct values, while target genes register Ct?values above 30.

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

01 qPCR program

A. Use a three-step instead of a two-step program.

B. Increase extension time to improve amplification efficiency.

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 genes nor interferes with the assay to minimize the?loss of target genes due to adhesion to pipette tips or tube walls during liquid handling.?

03 Reaction system

A. Template

Poor template purity, presence of inhibitors, template degradation, and insufficient input can all compromise qPCR results.

Recommendation:

• RNA: Evaluate RNA integrity with agarose gel electrophoresis and purity with NanoDrop.?
• Reverse transcription: Increase RNA template input.
• cDNA: Increase cDNA input or use a lower cDNA dilution factor (in theory, each 10-fold dilution increases the 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. Primers

Non-specific amplification consumes primers and Taq 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%.

Make serial dilutions of the gene amplification product (at least three dilutions) and run qPCR. Obtain Ct?value-based 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. Reagents

The compositions of Taq polymerase, buffers, Mg2+, and fluorescent dyes vary across different qPCR kits, affecting specificity, sensitivity, and amplification efficiency.

Recommendation:

Use kits specially designed for the detection of low-expression genes. Vazyme’s newly improved Taq Pro Universal SYBR qPCR Master Mix (Vazyme #Q712) is perfectly suited for enhancing your detection of low-expression genes.?Q712 demonstrates higher sensitivity for low-abundance and other challenging-to-amplify samples; it also offers high specificity and reduced viscosity. As of this year, more than 725 units of this product have been sold all of over the world.

Q712 Upgraded 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 Other?product?before upgrading, Vazyme #Q712 has a lower Ct?value at the limit of detection (the sixth dilution), exhibiting higher sensitivity.

• 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 a commercially available similar product from Supplier A. Results show that both kits yield detection rates predicted by the Poisson distribution. Vazyme #Q712 has a detection rate of 75%, superior to that of Supplier A’s product.

To learn more information or place an order online, please visit >>https://www.vazymeglobal.com/product-center/dye-based-qpcr/taq-pro-universal-sybr-qpcr-master-mix?channel=SM2420

*All rights reserved. All the statistics comes from Vazyme laboratory.