Selection and validation of reference genes for RT-qPCR analysis of different organs at various development stages in Caragana intermedia

Jinhua Liu; Chuang Yang; Mingzhu Bai; Feng Yan; Haiying Qin; Ruigang Wang; Yongqing Wan; Guojing Li

doi:10.1515/biol-2022-0463

Selection and validation of reference genes for RT-qPCR analysis of different organs at various development stages in Caragana intermedia

Open Life Sci. 2022 Sep 14;17(1):1155-1164. doi: 10.1515/biol-2022-0463. eCollection 2022.

Authors

Jinhua Liu¹, Chuang Yang¹, Mingzhu Bai¹, Feng Yan², Haiying Qin¹, Ruigang Wang^{1

3

4}, Yongqing Wan¹, Guojing Li¹

Affiliations

¹ College of Life Sciences, Inner Mongolia Key Laboratory of Plant Stress Physiology and Molecular Biology, Inner Mongolia Agricultural University, Hohhot 010018, P.R. China.
² Ordos Forestry and Grassland Development Center, Ordos 017010, P.R. China.
³ Inner Mongolia Enterprise Key Laboratory of Tree Breeding, Mengshu Ecological Construction Group Co., Ltd., Hohhot 011517, P.R. China.
⁴ Inner Mongolia Engineering Research Center for Plant Gene Resources Mining and Molecular Breeding, Inner Mongolia Agricultural University, Hohhot 010021, P.R. China.

Abstract

Reverse transcription quantitative PCR (RT-qPCR) is a technique widely used to investigate the expression of genes. An appropriate reference gene (RG) is essential for RT-qPCR analysis to obtain accurate and reliable results. Caragana intermedia plays an important role in afforestation as a bush. However, due to the lack of appropriate RGs, the research on development-related genes is limited. In this study, the selection for suitable RGs of different organs at various development stages to normalize the results of RT-qPCR about development-related genes was performed. To test the expression stability across all samples, we used the software algorithms such as geNorm, NormFinder, BestKeeper, and RefFinder to evaluate all the candidate RGs. Our results showed that CiEF1α was the most stable RG with little fluctuation among all samples. In addition, CiGAPDH in roots, CiSKIP1 in stems and leaves, and CiEF1α in different organs were selected as the most stable RGs. To confirm the applicability of the most stable RGs, the relative expression of CiWRKY17 was normalized using different candidate RGs. Taken together, our research laid a foundation for the study of development-related genes in C. intermedia.

Keywords: Caragana intermedia; RT-qPCR; development stage; reference gene.