Selection and validation of optimal housekeeping gene through RNA-sequence and qRT-PCR analysis in rat testis tissues and Sertoli cells under mangancism and Coridius chinensis extracts rescue

Selección y validación del gen constitutivo óptimo mediante análisis de transcriptoma (RNA-seq) y qRT-PCR en tejidos testiculares y células de Sertoli de rata bajo manganismo y tratamiento con extractos de Coridius chinensis

Background: Testicular tissues and cells undergoing injury or therapeutic interventions often exhibit significant changes in functional and housekeeping gene (HKG) expression. Using unstable HKGs can cause inaccurate quantification of target gene expression in quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Therefore, identifying reliable HKGs is critical for accurately assessing functional gene expression in studies of testicular injury and therapeutic interventions. Methods: In this study, 17 candidate HKGs were selected based on RNA-seq data and previous reports. The cycle threshold (Ct) values of these genes were determined by qRT-PCR in testicular tissue and Sertoli cells from the control (Ctrl) group, manganese ion (Mn²⁺) group, and Mn²⁺ combined with Coridius chinensis extract (Mn²⁺ + CcE) group. Expression stability was evaluated using four algorithms (geNorm, Normfinder, Bestkeeper, and Delta Ct), and comprehensively assessed by RefFinder and ComprFinder. The three most stable HKGs were validated by normalization to specific marker genes for spermatogonia and Sertoli cells (CASPASE3 (Cysteine-dependent aspartate-specific protease-3), DDX4 (DEAD box polypeptide 4), SOX9 (SRY-box transcription factor 9), and OCCLUDIN (Occludin)). Results: PPIA (Peptidylprolyl Isomerase A) exhibited the lowest Ct values in both testicular tissues and related cells. In testicular tissues, YBX1 (Y-box binding protein 1) and PPIA had the lowest Ct values across different treatment groups, while UBB (Ubiquitin B) and TUBULIN (tubulin) exhibited the highest Ct values. In Sertoli cells, PPIA and ACTB (Actin Beta) were identified as the most stable HKGs under various culture conditions. However, classical HKGs, such as TUBULIN and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) displayed unstable expression, indicating their unsuitability for target gene normalization. Conclusions: Our study demonstrates that PPIA may serve as the optimal HKG in rat testicular tissues and Sertoli cells under various pathological conditions, providing valuable reference data for functional gene expression studies related to testicular diseases in mammals.

Resumen
Antecedentes: Los tejidos y células testiculares sometidos a lesiones o intervenciones terapéuticas suelen presentar cambios significativos en la expresión de genes clave, incluidos los genes funcionales y los genes constitutivos (HKGs, por sus siglas en inglés). El uso de HKGs inestables puede conducir a una cuantificación errónea de la expresión génica objetivo en los análisis de PCR cuantitativa en tiempo real (qRT-PCR). Por lo tanto, la identificación de HKGs confiables es crucial para evaluar con precisión la expresión génica funcional en estudios relacionados con lesiones testiculares e intervenciones terapéuticas. Métodos: En este estudio se seleccionaron inicialmente 17 genes constitutivos candidatos basándose en datos de secuenciación de ARN (RNA-seq) y en informes previos. Los valores del umbral de ciclo (Ct) de estos genes se determinaron mediante qRT-PCR en tejido testicular y células de Sertoli de los grupos control (Ctrl), con ion manganeso (Mn²⁺) y con Mn²⁺ combinado con extracto de Coridius chinensis (Mn²⁺ + CcE). La estabilidad de expresión de estos genes candidatos fue evaluada mediante cuatro algoritmos (geNorm, NormFinder, BestKeeper y Delta Ct), y analizada de forma integral con RefFinder y ComprFinder. Los tres HKGs más estables fueron validados mediante su normalización con respecto a genes marcadores específicos de espermatogonias y células de Sertoli (CASPASE3 (Cysteine-dependent aspartate-specific protease-3), DDX4 (DEAD box polypeptide 4), SOX9 (SRY-box transcription factor 9) y OCCLUDIN (Occludin)). Resultados: PPIA (Peptidylprolyl Isomerase A) mostró los valores de Ct más bajos tanto en los tejidos testiculares como en las células asociadas. En los tejidos testiculares, YBX1 (Y-box binding protein 1) y PPIA como los valores de Ct más bajos entre los distintos grupos de tratamiento, mientras que UBB (Ubiquitin B) y TUBULINA (tubulin) presentaron los valores de Ct más elevados. En las células de Sertoli, PPIA y ACTB (Actin Beta) fueron identificados como los HKGs más estables bajo diversas condiciones de cultivo según la evaluación integral. Sin embargo, HKGs clásicos como TUBULINA y GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) mostraron una expresión inestable, lo que indica que no son adecuados para la normalización de genes diana. Conclusiones: Nuestro estudio demuestra que PPIA podría considerarse el único HKG óptimo en tejidos testiculares y células de Sertoli de rata bajo diversas condiciones patológicas, proporcionando datos de referencia valiosos para la selección de HKGs en estudios de expresión génica funcional relacionados con enfermedades testiculares y tratamientos en mamíferos.

Testis; Sertoli cell; Transcriptome; Housekeeping gene; Manganism; Coridius chinensis

Palabras Clave
Testículo; Célula de Sertoli; Transcriptoma; Gen constitutivo; Manganismo; Coridius chinensis

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