Reference gene selection for transcriptional profiling in Cryptocercus punctulatus, an evolutionary link between Isoptera and Blattodea

Zhen Li; Xiangrui Li; Qingwen Zhang; Ling Yuan; Xuguo Zhou

doi:10.1038/s41598-020-79030-6

Reference gene selection for transcriptional profiling in Cryptocercus punctulatus, an evolutionary link between Isoptera and Blattodea

Zhen Li, Xiangrui Li, Qingwen Zhang, Ling Yuan, Xuguo Zhou

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

Abstract

The subsocial life style and wood-feeding capability of Cryptocercus gives us an evolutionary key to unlock some outstanding questions in biology. With the advent of the Genomics Era, there is an unprecedented opportunity to address the evolution of eusociality and the acquisition of lignocellulases at the genetic level. However, to quantify gene expression, an appropriate normalization strategy is warranted to control for the non-specific variations among samples across different experimental conditions. To search for the internal references, 10 housekeeping genes from a gut transcriptome of a wood-feeding cockroach, Cryptocercus punctulatus, were selected as the candidates for the RT-qPCR analysis. The expression profiles of these candidates, including ACT, EF1α, GAPDH, HSP60, HSP70, αTUB, UBC, RPS18, ATPase and GST, were analyzed using a panel of analytical tools, including geNorm, NormFinder, BestKeeper, and comparative ΔC_T method. RefFinder, a comprehensive ranking system integrating all four above-mentioned algorithms, rated ACT as the most stable reference gene for different developmental stages and tissues. Expression analysis of the target genes, Hex-1 and Cell-1, using the most or the least appropriate reference genes and a single or multiple normalizers signified this research. Our finding is the first step toward establishing a standardized RT-qPCR analysis in Cryptocercus.

Original language	English
Article number	22169
Journal	Scientific Reports
Volume	10
Issue number	1
DOIs	https://doi.org/10.1038/s41598-020-79030-6
State	Published - Dec 2020

Bibliographical note

Funding Information:
Authors are grateful to Christine Nalepa (North Carolina State University) for providing C. punctulatus and for her comments on an earlier draft. Authors are also thankful to anonymous reviewers for their constructive comments and suggestions. This research was supported by a hatch project from USDA-NIFA (Accession No. 0220839; Project No. KY008053) and a pilot project from Kentucky Tobacco Research and Development Center (KTRDC), University of Kentucky. The information reported in this paper is part of a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. These agencies had no role in study design, data collection/analysis, manuscript preparation, or the decision to publish.

Publisher Copyright:
© 2020, The Author(s).

ASJC Scopus subject areas

General

Access to Document

10.1038/s41598-020-79030-6

Cite this

@article{2559311a27344ce486c0c0e5e5a8a4e4,

title = "Reference gene selection for transcriptional profiling in Cryptocercus punctulatus, an evolutionary link between Isoptera and Blattodea",

abstract = "The subsocial life style and wood-feeding capability of Cryptocercus gives us an evolutionary key to unlock some outstanding questions in biology. With the advent of the Genomics Era, there is an unprecedented opportunity to address the evolution of eusociality and the acquisition of lignocellulases at the genetic level. However, to quantify gene expression, an appropriate normalization strategy is warranted to control for the non-specific variations among samples across different experimental conditions. To search for the internal references, 10 housekeeping genes from a gut transcriptome of a wood-feeding cockroach, Cryptocercus punctulatus, were selected as the candidates for the RT-qPCR analysis. The expression profiles of these candidates, including ACT, EF1α, GAPDH, HSP60, HSP70, αTUB, UBC, RPS18, ATPase and GST, were analyzed using a panel of analytical tools, including geNorm, NormFinder, BestKeeper, and comparative ΔCT method. RefFinder, a comprehensive ranking system integrating all four above-mentioned algorithms, rated ACT as the most stable reference gene for different developmental stages and tissues. Expression analysis of the target genes, Hex-1 and Cell-1, using the most or the least appropriate reference genes and a single or multiple normalizers signified this research. Our finding is the first step toward establishing a standardized RT-qPCR analysis in Cryptocercus.",

author = "Zhen Li and Xiangrui Li and Qingwen Zhang and Ling Yuan and Xuguo Zhou",

note = "Funding Information: Authors are grateful to Christine Nalepa (North Carolina State University) for providing C. punctulatus and for her comments on an earlier draft. Authors are also thankful to anonymous reviewers for their constructive comments and suggestions. This research was supported by a hatch project from USDA-NIFA (Accession No. 0220839; Project No. KY008053) and a pilot project from Kentucky Tobacco Research and Development Center (KTRDC), University of Kentucky. The information reported in this paper is part of a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. These agencies had no role in study design, data collection/analysis, manuscript preparation, or the decision to publish. Publisher Copyright: {\textcopyright} 2020, The Author(s).",

year = "2020",

month = dec,

doi = "10.1038/s41598-020-79030-6",

language = "English",

volume = "10",

number = "1",

}

TY - JOUR

T1 - Reference gene selection for transcriptional profiling in Cryptocercus punctulatus, an evolutionary link between Isoptera and Blattodea

AU - Li, Zhen

AU - Li, Xiangrui

AU - Zhang, Qingwen

AU - Yuan, Ling

AU - Zhou, Xuguo

N1 - Funding Information: Authors are grateful to Christine Nalepa (North Carolina State University) for providing C. punctulatus and for her comments on an earlier draft. Authors are also thankful to anonymous reviewers for their constructive comments and suggestions. This research was supported by a hatch project from USDA-NIFA (Accession No. 0220839; Project No. KY008053) and a pilot project from Kentucky Tobacco Research and Development Center (KTRDC), University of Kentucky. The information reported in this paper is part of a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. These agencies had no role in study design, data collection/analysis, manuscript preparation, or the decision to publish. Publisher Copyright: © 2020, The Author(s).

PY - 2020/12

Y1 - 2020/12

N2 - The subsocial life style and wood-feeding capability of Cryptocercus gives us an evolutionary key to unlock some outstanding questions in biology. With the advent of the Genomics Era, there is an unprecedented opportunity to address the evolution of eusociality and the acquisition of lignocellulases at the genetic level. However, to quantify gene expression, an appropriate normalization strategy is warranted to control for the non-specific variations among samples across different experimental conditions. To search for the internal references, 10 housekeeping genes from a gut transcriptome of a wood-feeding cockroach, Cryptocercus punctulatus, were selected as the candidates for the RT-qPCR analysis. The expression profiles of these candidates, including ACT, EF1α, GAPDH, HSP60, HSP70, αTUB, UBC, RPS18, ATPase and GST, were analyzed using a panel of analytical tools, including geNorm, NormFinder, BestKeeper, and comparative ΔCT method. RefFinder, a comprehensive ranking system integrating all four above-mentioned algorithms, rated ACT as the most stable reference gene for different developmental stages and tissues. Expression analysis of the target genes, Hex-1 and Cell-1, using the most or the least appropriate reference genes and a single or multiple normalizers signified this research. Our finding is the first step toward establishing a standardized RT-qPCR analysis in Cryptocercus.

AB - The subsocial life style and wood-feeding capability of Cryptocercus gives us an evolutionary key to unlock some outstanding questions in biology. With the advent of the Genomics Era, there is an unprecedented opportunity to address the evolution of eusociality and the acquisition of lignocellulases at the genetic level. However, to quantify gene expression, an appropriate normalization strategy is warranted to control for the non-specific variations among samples across different experimental conditions. To search for the internal references, 10 housekeeping genes from a gut transcriptome of a wood-feeding cockroach, Cryptocercus punctulatus, were selected as the candidates for the RT-qPCR analysis. The expression profiles of these candidates, including ACT, EF1α, GAPDH, HSP60, HSP70, αTUB, UBC, RPS18, ATPase and GST, were analyzed using a panel of analytical tools, including geNorm, NormFinder, BestKeeper, and comparative ΔCT method. RefFinder, a comprehensive ranking system integrating all four above-mentioned algorithms, rated ACT as the most stable reference gene for different developmental stages and tissues. Expression analysis of the target genes, Hex-1 and Cell-1, using the most or the least appropriate reference genes and a single or multiple normalizers signified this research. Our finding is the first step toward establishing a standardized RT-qPCR analysis in Cryptocercus.

UR - http://www.scopus.com/inward/record.url?scp=85097671420&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85097671420&partnerID=8YFLogxK

U2 - 10.1038/s41598-020-79030-6

DO - 10.1038/s41598-020-79030-6

M3 - Article

C2 - 33335167

AN - SCOPUS:85097671420

VL - 10

IS - 1

M1 - 22169

ER -

Reference gene selection for transcriptional profiling in Cryptocercus punctulatus, an evolutionary link between Isoptera and Blattodea

Abstract

Bibliographical note

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this