Cargando…
Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT-qPCR experiments. However, the stability of housekeeping genes in spinach (Spinacia olerace...
Autores principales: | , , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Hindawi
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875621/ https://www.ncbi.nlm.nih.gov/pubmed/33623781 http://dx.doi.org/10.1155/2021/4853632 |
_version_ | 1783649796650696704 |
---|---|
author | Xie, Hao Li, Bo Chang, Yu Hou, Xiaoyan Zhang, Yue Guo, Siyi Miao, Yuchen Wang, Quanhua Chen, Sixue Su, Yinghua Li, Ying Dai, Shaojun |
author_facet | Xie, Hao Li, Bo Chang, Yu Hou, Xiaoyan Zhang, Yue Guo, Siyi Miao, Yuchen Wang, Quanhua Chen, Sixue Su, Yinghua Li, Ying Dai, Shaojun |
author_sort | Xie, Hao |
collection | PubMed |
description | Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT-qPCR experiments. However, the stability of housekeeping genes in spinach (Spinacia oleracea) under various abiotic stresses is unclear. Evaluating the stability of candidate genes and determining the optimal gene(s) for normalization of gene expression in spinach are necessary to investigate the gene expression patterns during development and stress response. In this study, ten housekeeping genes, 18S ribosomal RNA (18S rRNA), actin, ADP ribosylation factor (ARF), cytochrome c oxidase subunit 5C (COX), cyclophilin (CYP), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (H3), 50S ribosomal protein L2 (RPL2), and tubulin alpha chain (TUBα) from spinach, were selected as candidates in roots, stems, leaves, flowers, and seedlings in response to high temperature, CdCl(2), NaCl, NaHCO(3), and Na(2)CO(3) stresses. The expression of these genes was quantified by RT-qPCR and evaluated by NormFinder, BestKeeper, and geNorm. 18S rRNA, actin, ARF, COX, CYP, EF1α, GAPDH, H3, and RPL2 were detected as optimal reference genes for gene expression analysis of different organs and stress responses. The results were further confirmed by the expression pattern normalized with different reference genes of two heat-responsive genes. Here, we optimized the detection method of the gene expression pattern in spinach. Our results provide the optimal candidate reference genes which were crucial for RT-qPCR analysis. |
format | Online Article Text |
id | pubmed-7875621 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Hindawi |
record_format | MEDLINE/PubMed |
spelling | pubmed-78756212021-02-22 Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress Xie, Hao Li, Bo Chang, Yu Hou, Xiaoyan Zhang, Yue Guo, Siyi Miao, Yuchen Wang, Quanhua Chen, Sixue Su, Yinghua Li, Ying Dai, Shaojun Biomed Res Int Research Article Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT-qPCR experiments. However, the stability of housekeeping genes in spinach (Spinacia oleracea) under various abiotic stresses is unclear. Evaluating the stability of candidate genes and determining the optimal gene(s) for normalization of gene expression in spinach are necessary to investigate the gene expression patterns during development and stress response. In this study, ten housekeeping genes, 18S ribosomal RNA (18S rRNA), actin, ADP ribosylation factor (ARF), cytochrome c oxidase subunit 5C (COX), cyclophilin (CYP), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (H3), 50S ribosomal protein L2 (RPL2), and tubulin alpha chain (TUBα) from spinach, were selected as candidates in roots, stems, leaves, flowers, and seedlings in response to high temperature, CdCl(2), NaCl, NaHCO(3), and Na(2)CO(3) stresses. The expression of these genes was quantified by RT-qPCR and evaluated by NormFinder, BestKeeper, and geNorm. 18S rRNA, actin, ARF, COX, CYP, EF1α, GAPDH, H3, and RPL2 were detected as optimal reference genes for gene expression analysis of different organs and stress responses. The results were further confirmed by the expression pattern normalized with different reference genes of two heat-responsive genes. Here, we optimized the detection method of the gene expression pattern in spinach. Our results provide the optimal candidate reference genes which were crucial for RT-qPCR analysis. Hindawi 2021-02-03 /pmc/articles/PMC7875621/ /pubmed/33623781 http://dx.doi.org/10.1155/2021/4853632 Text en Copyright © 2021 Hao Xie et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Xie, Hao Li, Bo Chang, Yu Hou, Xiaoyan Zhang, Yue Guo, Siyi Miao, Yuchen Wang, Quanhua Chen, Sixue Su, Yinghua Li, Ying Dai, Shaojun Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress |
title | Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress |
title_full | Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress |
title_fullStr | Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress |
title_full_unstemmed | Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress |
title_short | Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress |
title_sort | selection and validation of reference genes for rt-qpcr analysis in spinacia oleracea under abiotic stress |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875621/ https://www.ncbi.nlm.nih.gov/pubmed/33623781 http://dx.doi.org/10.1155/2021/4853632 |
work_keys_str_mv | AT xiehao selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress AT libo selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress AT changyu selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress AT houxiaoyan selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress AT zhangyue selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress AT guosiyi selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress AT miaoyuchen selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress AT wangquanhua selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress AT chensixue selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress AT suyinghua selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress AT liying selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress AT daishaojun selectionandvalidationofreferencegenesforrtqpcranalysisinspinaciaoleraceaunderabioticstress |