How to Choose Reference Genes for Relative Quantification Experiments?

Relative quantification is suitable for most gene expression studies, which can analyze the upregulation or downregulation of target gene expression levels in calibrated (normal) samples and one or more experimental samples. This method does not require precise determination of copy number, but focuses on the changes in the relative expression level of the target gene. The ΔΔCt method is a very common relative quantitative analysis method, which can be used to compare the results of experimental samples including calibrators (such as untreated or wild-type samples) and standards (such as housekeeping gene expression). With this method, the Ct of the target gene in the same two samples can be compared according to the Cq of the standard (normal) gene in the test sample and the calibrated sample, and the obtained ΔΔCt value can be used to determine the fold difference in expression.

Relative quantification experiments are commonly used to:

Compare the expression levels of genes in different tissues;

Compare the expression levels of genes in treated and untreated samples;

Compare the expression levels of genes of interest under different genetic backgrounds;

Analyze the temporal changes of gene expression under specific treatments.

The first step in relative quantification experiments is to select appropriate reference genes, which is crucial to ensure the accuracy and reliability of qPCR (quantitative real-time PCR) experimental results. Common reference genes include GAPDH, β-actin, 18S rRNA, etc., but the specific selection of one or more reference genes depends on the experimental conditions, sample types and the stability of target gene expression levels.

01. GAPDH (Glyceraldehyde-3-phosphate Dehydrogenase)

Advantages

Widely used: GAPDH is one of the most commonly used reference genes in qPCR experiments because its expression is relatively stable in most tissues and cells.

Wide applicability: GAPDH usually shows a relatively consistent expression level in various cell types and experimental conditions.

Disadvantages

Expression stability issues: In some cases, such as cell proliferation, apoptosis or specific treatment conditions (such as oxidative stress or high glucose environment), the expression of GAPDH may fluctuate, so it is not suitable as a reference gene in these cases.

Related to metabolic activity: GAPDH is involved in the glycolysis process, and changes in metabolic status may affect its expression.

02. β-actin (Beta-Actin)

Advantages

Cytoskeletal gene: β-actin encodes cytoskeletal proteins, which are highly expressed and usually relatively stable in most cells.

Wide applicability: Similar to GAPDH, β-actin is also a commonly used reference gene, widely applicable to different types of cells and tissues.

Disadvantages

Expression fluctuation: The expression of β-actin may change during cell division, migration or remodeling. Under certain experimental conditions, such as in cancer cells or immune cells, its expression may also be unstable.

Presence of pseudogenes: β-actin has pseudogenes (non-coding similar sequences), which may lead to non-specific amplification in qPCR.

03. Other Reference Genes

18S rRNA: As ribosomal RNA, 18S rRNA is usually highly expressed and relatively stable under different conditions, suitable for experiments with a large number of RNA samples. However, due to its high expression level, sample dilution may be required to keep it in the same linear range as the target gene.

HPRT1 (Hypoxanthine Phosphoribosyltransferase 1): It is often used in cancer research and gene expression research, and its expression is relatively stable, but it may fluctuate in some tissues.

RPLP0 (Ribosomal Protein Large Subunit P0): It encodes ribosomal proteins and is another reference gene with good stability, widely used under different experimental conditions.

04. Principles for Selecting Reference Genes

Expression stability: The expression of reference genes should be stable under all experimental conditions. Ideally, the expression level of the reference gene is not affected by experimental treatment, cell state or tissue type.

Adapt to sample types: Select appropriate reference genes according to the cell type or tissue you are studying. For example, GAPDH and β-actin are stably expressed in most mammalian cells, but may not be suitable under certain special conditions.

Use of multiple references: In many cases, normalization using multiple reference genes can improve the accuracy and reliability of data. Software such as GeNorm or NormFinder can be used to evaluate the stability of multiple candidate reference genes, thereby selecting the best combination of references.

Application Case Sharing

Relative Quantitative Analysis of Gene Expression Using Archimed (△△Ct Method)

Below, we will take a real experiment as an example to show the experimental results of relative quantitative analysis of gene expression using Archimed quantitative PCR. In this experiment, the HELA cell line was used as the experimental material, and FOXCL2-M8 and FOXCL2-M10 plasmids were transfected into the HELA cell line. The purpose was to study the effects of the two plasmids on the expression of three genes (IL2A, star-qpcr1 and IER3) in a certain signaling pathway, and GAPDH was selected as the reference gene.

Experimental Results

According to the analysis of experimental results, the four genes including the reference gene showed good amplification repeatability and specificity on Archimed X6, with the SD of CT values all ≤ 0.1; after transfecting the two plasmids into the HELA cell line, the expressions of IER3 and IL2A genes were decreased, while the expression of star-qpcr1 gene was upregulated.

Amplification Curve Results

Melting Curve Results

Gene Expression Difference Results

Summary

Routine experiments: If the sample types and experimental treatment conditions are routine, GAPDH or β-actin are usually the first choices.

Complex conditions: When dealing with complex experimental conditions or special sample types, it is recommended to test the stability of multiple reference genes and select the most stably expressed ones.

Use multiple references: To ensure the accuracy of qPCR data, especially in critical research, normalization using multiple reference genes is the best practice.

Ultimately, the selection of reference genes should be based on the specific circumstances of the experiment and verified experimentally before being applied to qPCR analysis.

Of course, the presentation of excellent relative quantification results depends not only on the selection of reference genes, but also on the performance of reagents and the choice of equipment, just like Rocgene's Archimed series QPCR instrument.

Archimed Series

Quantitative Real-Time PCR Instrument

The Archimed series quantitative real-time PCR instrument is the world's first time-resolved real-time fluorescent quantitative PCR system meticulously built by Rocgene's international senior technical team, drawing on the essence of the development of quantitative PCR technology. Based on the new optical path system of Fresnel lens, patented time-resolved signal acquisition technology and unique temperature control technology, Archimed has higher detection sensitivity, more excellent temperature control accuracy and uniformity, more convenient operation process, and more comprehensive analysis functions. At the same time, based on the global vision of product design concept and manufacturing process, Archimed is endowed with excellent quality of international standards.