Reference gene evaluation for digital PCR; Applications for RNA biomarker testing in cervical precancer

Area of Science:

• Molecular Biology
• Gynecologic Oncology
• Biomarker Discovery

Background:

• Persistent high-risk human papillomavirus (hrHPV) infection is the primary cause of cervical cancer.
• Cervical screening reduces incidence, but novel tests are needed to distinguish clinically significant HPV infections from asymptomatic cases.
• Digital PCR (dPCR) offers precise quantification for mRNA biomarker validation.

Purpose of the Study:

• To evaluate the suitability of eight reference genes for normalizing dPCR gene expression data in cervical intraepithelial neoplasia (CIN) samples.
• To identify stable reference genes for accurate biomarker quantification in liquid-based cytology (LBC).

Main Methods:

• Assessed eight candidate reference genes (ACTB, GAPDH, RPP30, HPRT1, HMBS, MT-ATP6, UBE2D2, GUSB) in CIN1 and CIN3 LBC samples.
• Utilized geNorm and NormFinder algorithms to compare reference gene stability.
• Analyzed gene expression data for normalization accuracy in dPCR assays.

Main Results:

• geNorm analysis identified GAPDH, ACTB, GUSB, and MT-ATP6 as the top four performing genes.
• GAPDH and ACTB showed highest stability but also very high expression levels, potentially unsuitable for low-expressed biomarkers.
• GUSB and HMBS were identified as a stable pair with suitable expression levels for dPCR normalization.

Conclusions:

• The study recommends GUSB and HMBS as a reliable reference gene pair for normalizing dPCR data in cervical disease LBC samples.
• Accurate normalization using stable reference genes is essential for validating mRNA biomarkers in cervical cancer research.
• This finding aids in minimizing unnecessary diagnoses and treatments by improving the precision of HPV biomarker detection.