The pseudogene problem and RT-qPCR data normalization; SYMPK: a suitable reference gene for papillary thyroid...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Quantitative real-time PCR (RT-qPCR) accuracy depends on robust standardization.
• Data normalization using reference genes is crucial but can be compromised by pseudogenes.
• Papillary thyroid carcinoma (PTC) research necessitates reliable gene expression analysis.

Purpose of the Study:

• To identify and validate stable reference genes for RT-qPCR in papillary thyroid carcinoma (PTC) tissues.
• To address the challenge of pseudogene interference in gene expression normalization.
• To enhance the accuracy of RT-qPCR results in PTC studies.

Main Methods:

• Selection of eight candidate reference genes based on literature and microarray data analysis.
• RNA extraction, cDNA synthesis, and RT-qPCR amplification using exon-junction or intron-spanning primers.
• Statistical analysis using Microsoft Excel, NormFinder, and BestKeeper to assess gene stability.

Main Results:

• SYMPK exhibited the lowest coefficient of variation (CqCV%) and maximum fold change (MFC) in PTC tissues.
• NormFinder identified SYMPK and ACTB as the optimal reference gene combination.
• BestKeeper analysis consistently ranked SYMPK as the most stable reference gene in both normal and PTC tissues.

Conclusions:

• SYMPK is recommended as a highly stable reference gene for accurate RT-qPCR data normalization in papillary thyroid carcinoma.
• The use of SYMPK effectively overcomes pseudogene-related confounding factors.
• Validated reference genes improve the reliability of gene expression studies in cancer research.