Relative quantification of BCL2 mRNA for diagnostic usage needs stable uncontrolled genes as reference

Area of Science:

• Molecular biology
• Oncology
• Genetics

Background:

• Dysregulation of the BCL2 gene is a key factor in the pathophysiology of hematological malignancies.
• Accurate relative quantification of BCL2 mRNA is essential for implementing effective treatment strategies.
• The selection of appropriate reference genes is critical for reliable gene expression analysis in qPCR.

Purpose of the Study:

• To identify and validate novel reference genes for accurate BCL2 mRNA quantification in hematological malignancies.
• To compare the stability and performance of newly identified reference genes against commonly used ones.
• To establish a more reliable method for BCL2 mRNA expression analysis in cancer diagnostics.

Main Methods:

• Selection of potential reference genes through analysis of RNA-seq datasets from hematological malignancies, focusing on gene expression variation, GO annotations, and chromosomal proximity to disease translocations.
• Experimental validation of selected genes across diverse hematological malignancy samples.
• Stability assessment of reference genes using geNorm, NormFinder, BestKeeper, and RefFinder algorithms.
• Systematic review to identify 43 commonly used reference genes from existing literature.
• Quantitative PCR (qPCR) analysis of BCL2 mRNA levels, normalized by novel reference genes and GAPDH, followed by comparative analysis.

Main Results:

• PTCD2, PPP1R3B, and FBXW9 were identified as the most consistently unregulated genes across lymph-node, bone marrow, and peripheral blood mononuclear cell (PBMC) samples.
• These novel reference genes demonstrated superior stability compared to commonly used reference genes.
• Normalization with the novel reference genes revealed consistently higher BCL2 mRNA expression in hematological malignancy patients than normalization with GAPDH.
• The identified reference genes are potentially applicable across various qPCR platforms (e.g., Taqman probes) and model systems, including cell lines and rodent models.

Conclusions:

• The novel reference genes PTCD2, PPP1R3B, and FBXW9 offer a more reliable basis for BCL2 mRNA quantification in hematological malignancies.
• Accurate BCL2 mRNA measurement, normalized by stable reference genes, is vital for its potential use in molecular diagnostics.
• Careful selection of reference genes is paramount, especially in diagnostic settings lacking healthy-normal controls, to ensure the validity of qPCR-based biomarker quantification.