Validation of reference genes for quantitative PCR in the forest pest, Ips calligraphus

Mary Wallace, Lynne K. Rieske

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


The six-spined ips, Ips calligraphus, is a North American bark beetle that can exploit most eastern North American Pinus species and can cause mortality. Biotic and abiotic disturbances weaken trees, creating breeding substrate that promotes rapid population growth. Management historically relied on silvicultural practices, but as forests become increasingly stressed, innovative management is needed. Manipulation of the cellular RNA interference (RNAi) pathway to induce gene silencing is an emerging means of insect suppression, and is effective for some bark beetles. Quantitative PCR (qPCR) is a powerful tool for analysis of gene expression, and is essential for examining RNAi. To compare gene expression among individuals, stably expressed reference genes must be validated for qPCR. We evaluated six candidate reference genes (18s, 16s, 28s, ef1a, cad, coi) for stability under biotic (beetle sex, developmental stage, and host plant), and abiotic (temperature, photoperiod, and dsRNA exposure) conditions. We used the comprehensive RefFinder tool to compare stability rankings across four algorithms. These algorithms identified 18s, 16s, and 28s as the most stably expressed. Overall, 16s and 28s were selected as reference genes due to their stability and moderate expression levels, and can be used for I. calligraphus gene expression studies using qPCR, including those evaluating RNAi.

Original languageEnglish
Article number23523
JournalScientific Reports
Issue number1
StatePublished - Dec 2021

Bibliographical note

Funding Information:
The authors thank Jeffrey Eickwort, Holly Foster, Hannah Hollowell, and J. David Wallace for assistance obtaining beetles, and Zachary Bragg, Bethany Kyre, and Flávia Pampolini for laboratory assistance. This work was supported by funds provided by USDA APHIS AP20PPQS&T00C061, the University of Kentucky, and the Kentucky Agricultural Experiment Station under McIntire-Stennis 2351197000, and is published with the approval of the director.

Publisher Copyright:
© 2021, The Author(s).

ASJC Scopus subject areas

  • General


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