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Background <p>Accurate normalisation of quantitative gene expression data requires stably expressed reference genes. However, condition-specific suitable reference genes have not been systematically evaluated and identified in pink bollworm <i>Pectinophora gossypiella</i> (Lepidoptera: Gelechiidae), a significant cotton pest.</p> Methods and results <p>In this study, expression stability of eight candidate reference genes, namely <i>EF1</i>,<i> EF2</i>,<i> GAPDH</i>,<i> ACT5C1</i>,<i> α-TUB</i>,<i> β-TUB</i>,<i> RPL3 and RPL13</i>, was assessed under distinct experimental conditions, including developmental stages, tissues, temperature variations, growth regulators, Cry toxin exposure and RNAi. Expression stability was analysed using standard algorithms, ΔCt, BestKeeper, NormFinder, and geNorm. A comprehensive ranking was generated using RefFinder, integrating rankings from all analytical approaches. Additionally, GrayNorm algorithm was employed to identify condition-specific optimal gene combinations. The stability rankings varied depending on experimental conditions. RefFinder identified <i>ACT5C1</i> and <i>RPL13</i> as the most stable reference genes across developmental stages and tissues, respectively. Under stress conditions taken together, it ranked <i>α-TUB</i> as the best performing reference gene. Meanwhile, GrayNorm identified ‘<i>ACT5C1</i>’, <i>‘ACT5C1 + EF2’</i> and <i>‘ACT5C1 + EF1 + GAPDH’</i> for normalizing gene expression data in developmental stages, while recognising <i>‘EF2’</i>, ‘<i>ACT5C1 + GAPDH’</i>, ‘<i>ACT5C1 + EF2 + GAPDH</i>’ in tissues, and ‘<i>RPL13</i>’, ‘<i>ACT5C1</i> + <i>RPL13</i>’, ‘<i>ACT5C1 + RPL3 + RPL13</i>’ under stress conditions, respectively.</p> Conclusions <p>Based on the consensus of all algorithms tested, we propose <i>ACT5C1</i>, <i>EF2</i> and <i>GAPDH</i> as the optimal gene combination for gene expression studies across developmental stages and tissues, while <i>ACT5C1</i>, <i>RPL3</i> and <i>RPL13</i> for studies under diverse stress conditions in pink bollworm. This study reports the first comprehensive evaluation of reference genes providing critical resources for accurate gene expression analysis under diverse experimental conditions in pink bollworm.</p> Graphical abstract <p></p>

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Identification of condition-specific optimal reference genes for qPCR data normalisation in pink bollworm Pectinophora gossypiella (Saunders)

  • 期刊论文

  • 发表日期：

    2026年6月30日

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  • Joy Das,
  • Rakesh Kumar,
  • K. P. Raghavendra ,
  • Suhas G. Karkute,
  • Vivek Shah,
  • Kishor U. Tribhuvan,
  • Rahul M. Phuke,
  • G. T. Behere,
  • Vinita Gotmare,
  • V. N. Waghmare

摘要

Background

Accurate normalisation of quantitative gene expression data requires stably expressed reference genes. However, condition-specific suitable reference genes have not been systematically evaluated and identified in pink bollworm Pectinophora gossypiella (Lepidoptera: Gelechiidae), a significant cotton pest.

Methods and results

In this study, expression stability of eight candidate reference genes, namely EF1, EF2, GAPDH, ACT5C1, α-TUB, β-TUB, RPL3 and RPL13, was assessed under distinct experimental conditions, including developmental stages, tissues, temperature variations, growth regulators, Cry toxin exposure and RNAi. Expression stability was analysed using standard algorithms, ΔCt, BestKeeper, NormFinder, and geNorm. A comprehensive ranking was generated using RefFinder, integrating rankings from all analytical approaches. Additionally, GrayNorm algorithm was employed to identify condition-specific optimal gene combinations. The stability rankings varied depending on experimental conditions. RefFinder identified ACT5C1 and RPL13 as the most stable reference genes across developmental stages and tissues, respectively. Under stress conditions taken together, it ranked α-TUB as the best performing reference gene. Meanwhile, GrayNorm identified ‘ACT5C1’, ‘ACT5C1 + EF2’ and ‘ACT5C1 + EF1 + GAPDH’ for normalizing gene expression data in developmental stages, while recognising ‘EF2’, ‘ACT5C1 + GAPDH’, ‘ACT5C1 + EF2 + GAPDH’ in tissues, and ‘RPL13’, ‘ACT5C1 + RPL13’, ‘ACT5C1 + RPL3 + RPL13’ under stress conditions, respectively.

Conclusions

Based on the consensus of all algorithms tested, we propose ACT5C1, EF2 and GAPDH as the optimal gene combination for gene expression studies across developmental stages and tissues, while ACT5C1, RPL3 and RPL13 for studies under diverse stress conditions in pink bollworm. This study reports the first comprehensive evaluation of reference genes providing critical resources for accurate gene expression analysis under diverse experimental conditions in pink bollworm.

Graphical abstract