A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Quantitative PCR (qPCR) relies on consistent standards for accurate gene expression measurement.
• Both endogenous and exogenous standards are used in qPCR, but their comparative reliability is not well-established.

Purpose of the Study:

• To compare the reliability of endogenous and exogenous standards for quantitative PCR.
• To determine if qPCR assays can accurately detect known dilutions of single-stranded cDNA using different standard types.

Main Methods:

• Tested exogenous recombinant DNA (VEGF-mimic plasmid) and endogenous GAPDH standards.
• Measured VEGF ss-cDNA copy numbers and GAPDH/VEGF signal ratios in 10x and 100x diluted samples.
• Evaluated the ability of each standard to detect known dilution factors in two cell lines (CaSki and U-251 MG).

Main Results:

• Exogenous standards accurately detected dilution factors (e.g., 10.3x and 135.0x for 10x and 100x dilutions).
• Endogenous GAPDH standards showed significant inaccuracies in detecting dilution factors (e.g., 2.7x and 8.0x).
• GAPDH mRNA copy numbers were ~1000 times higher than VEGF, indicating a high internal lockup ratio.

Conclusions:

• Abundant endogenous standards like GAPDH are unreliable for quantitative or semi-quantitative PCR due to high internal variation.
• Exogenous standard-based quantitative PCR is an accurate and reliable method for quantifying gene expression.
• Exogenous standards offer superior precision and consistency for gene expression analysis via qPCR.