Assessment of suitable reference genes for qRT-PCR analysis in Adelphocoris suturalis

Authors: LUO, JING - Huazhong Agricultural University; MA, CHAO - Huazhong Agricultural University; LI, ZHE - Huazhong Agricultural University; ZHU, BANGQIN - Huazhong Agricultural University; LEI, CHAOLIANG - Huazhong Agricultural University; JIN, SHUANGXIA - Huazhong Agricultural University; Hull, Joe; CHEN, LIZHEN - Huazhong Agricultural University

Submitted to: Journal of Integrative Agriculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/2/2018
Publication Date: 12/10/2018
Citation: Luo, J., Ma, C., Li, Z., Zhu, B., Lei, C., Jin, S., Hull, J.J., Chen, L. 2018. Assessment of suitable reference genes for qRT-PCR analysis in Adelphocoris suturalis. Journal of Integrative Agriculture. 17(12):2745-2757.

Interpretive Summary: Changes in mRNA transcript levels that reflect gene expression within specific spatio-temporal contexts and/or experimental conditions are frequently assessed using quantitative reverse transcription PCR (qRT-PCR). Advantages of this method include its sensitivity, specificity, reproducibility, and compatibility with high-throughput analyses. The fidelity of qRT-PCR, however, can be hampered by sample-to-sample variability. To overcome this limitation, the expression levels of genes of interest (i.e. targets) are normalized to reference genes that are stably expressed under the conditions being analyzed. Failure to identify reference genes appropriate for a particular condition can lead to incorrect interpretations and erroneous conclusions. Because there are no universal reference genes appropriate for each species, tissue, and/or condition, it is essential that a set of candidate reference genes be validated prior to assessing the transcript levels of target genes. To identify reference genes appropriate for assessing gene expression in the cotton pest species Adelphocoris suturalis, the transcript levels of 12 candidate genes were examined within the context of four biological conditions: insect developmental age and sex, adult tissue type, developmental stage and sex of metathoracic glands, and in response to dsRNA injection. Although candidate genes varied depending on the biological condition analyzed, a set of best-choice genes were identified for each condition. This study not only facilitates future studies assessing A. suturalis gene expression, but also provides insights into which genes in other pest species may be appropriate for qRT-PCR normalization.

Technical Abstract: Abstract - Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most commonly-used tool for measurement of gene expression, but its accuracy and reliability depend on appropriate data normalization with the use of one or more stable reference genes. Adelphocoris suturalis is one of the most destructive pests of cotton, but until recently knowledge of its underlying molecular physiology had been hindered by a lack of molecular resources. To facilitate research on this pest, we evaluated 12 common housekeeping genes studied in insects (GAPDH, ACT, ßACT, TBP, SDH, ßTUB, EF1', EF1a, EF1d, RPL32, RPS15, and RPL27) for their expression stability in A. suturalis when subjected to various experimental treatments, including three biotic (developmental stage and sex, tissue type, and metathoracic scent gland for varying developmental stages and sexes) and one abiotic (RNA interference injection) conditions. Four dedicated algorithms ('Ct method, geNorm, BestKeeper and NormFinder) were used to analyze gene expression stability. In addition, RefFinder provided an overall ranking of the stability/suitability of these candidates. This study is the first to provide a comprehensive list of suitable reference genes for gene expression analyses in A. suturalis, which can serve to facilitate transcript expression study of related biological processes in this and related species.