Defining stable reference genes in HIV latency reversal experiments

Area of Science:

• Virology
• Molecular Biology
• Immunology

Background:

• Cell-associated HIV RNA (ca-RNA) quantification is vital for assessing latency-reversing agents (LRAs) in HIV cure strategies.
• qPCR normalization typically uses reference genes (RGs), but their stability under LRA treatment is unverified.
• Biological variability and technical errors can affect normalization if RGs are not stable.

Purpose of the Study:

• To evaluate the stability of commonly used reference genes (RGs) in human PBMCs and CD4+ T cells after exposure to LRAs.
• To identify the most stable RGs for accurate quantification of HIV ca-RNA and host gene expression.
• To assess the impact of unstable RGs on experimental outcomes in LRA-treated cells.

Main Methods:

• Analyzed the expression stability of six reference genes (GAPDH, TBP, YWHAZ, UBE2D2, HPRT1, RPL27A) in human PBMCs and CD4+ T cells.
• Treated cells with LRAs to mimic experimental conditions.
• Quantified HIV ca-RNA and host gene expression using qPCR and assessed RG stability using established algorithms.

Main Results:

• LRA exposure significantly altered the expression stability of most tested RGs.
• TBP, UBE2D2, and RPL27A demonstrated the highest stability across all tested conditions.
• GAPDH exhibited the most variability, while TBP was generally the most stable.
• Using unstable RGs for normalization led to altered results in both host and HIV gene expression.

Conclusions:

• Reference gene stability is significantly compromised by LRA treatment.
• TBP, UBE2D2, and RPL27A are recommended as stable RGs for LRA studies.
• Accurate evaluation of LRA efficacy requires careful selection and validation of reference genes to avoid data misinterpretation.