Validation of Housekeeping Genes for Normalizing RNA Expression in Real-Time PCR in Tuberculomas and Peripheral Blood Mononuclear Cells for Pulmonary Tuberculosis Patients

Accurate normalization of qRT-PCR data in pulmonary tuberculosis (TB) research requires reference genes whose expression is invariant across clinically relevant matrices, yet no studies have addressed this in lesion tissue and blood concurrently. We assessed the expression stability of eight popular housekeeping genes-<i>ACTB</i>, <i>B2M</i>, <i>GAPDH</i>, <i>HPRT1</i>, <i>PPIA</i>, <i>RPL13A</i>, <i>UBC</i> and <i>YWHAZ</i>-in lung tuberculomas and peripheral blood mononuclear cells (PBMCs) from TB patients. Standardized extraction and amplification yielded Cq values that were ranked by geNorm, NormFinder, BestKeeper and comparative Delta CT, with consensus scores generated in RefFinder; and correlation analysis was conducted in order to select the most suitable genes to work collectively for future normalization. The consensus analysis placed <i>PPIA</i>, <i>YWHAZ</i> and <i>HPRT1</i> at the top, while <i>GAPDH</i> and <i>UBC</i> were the least stable. Our findings endorse a three-gene panel (<i>PPIA</i>, <i>YWHAZ</i>, <i>HPRT1</i>) for robust normalization of host gene-expression studies in both lesion tissue and PBMCs in pulmonary TB and highlight the necessity of context-specific reference-gene validation.