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Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses

Authors :
Zhongjun Gong
Jing Zhang
Qi Chen
Huiling Li
Ziqi Zhang
Yun Duan
Yueli Jiang
Tong Li
Jin Miao
Yuqing Wu
Source :
Insects, Vol 15, Iss 9, p 661 (2024)
Publication Year :
2024
Publisher :
MDPI AG, 2024.

Abstract

Holotrichia parallela is among the world’s most destructive pests. For accurate qPCR and gene expression studies, the selection of stable and appropriate reference genes is crucial. However, a thorough evaluation of potential reference genes for use in H. parallela research is lacking. In this study, 11 reference genes (GAPDH, RPL32, RPL7A, RPS18, RPL13a, RPL18, Actin, RPS7, RPS3, VATB,and EF1A) were evaluated under different biological conditions and environmental stresses. The stability of 11 potential reference gene transcripts was evaluated through various computational tools, including geNorm, BestKeeper, NormFinder, theΔCt method, and the RefFinder program. Under various developmental stages and RNAi conditions, RPL18 and RPL13a exhibited the greatest stability. RPL13a, RPL18, and RPL32 were the most stable genes in both male and female adults. Under differing tissue conditions, RPL13a and RPS3 stood out as the most reliable. Moreover, under varying photoperiod conditions, RPL13a, RPS3 and RPL32 were the most stable genes. Lastly, Actin and RPL13a were the most stable genes across different temperatures. These findings offer essential criteria for selecting suitable reference genes across diverse experimental settings, thereby establishing a solid basis for accurate gene expression studies in H. parallela using RT-qPCR.

Details

Language :
English
ISSN :
20754450
Volume :
15
Issue :
9
Database :
Directory of Open Access Journals
Journal :
Insects
Publication Type :
Academic Journal
Accession number :
edsdoj.37084ae6806b4b829ca3a5780b3e4200
Document Type :
article
Full Text :
https://doi.org/10.3390/insects15090661