IDENTIFICATION AND VALIDATION OF REFERENCE GENES FOR THE NORMALIZATION IN REAL-TIME RT-QPCR ON RICE AND RED RICE IN COMPETITION, UNDER DIFFERENT NITROGEN DOSES

BENEMANN, D.P.; NOHATO, A.M.; VARGAS, L.; AVILA, L.A; AGOSTINETTO, D.

doi:10.1590/S0100-83582017350100015

Acessibilidade / Reportar erro

Brasil

Español English

sumário « anterior atual seguinte »

Sumário

Article • Planta daninha 35 • 2017 • https://doi.org/10.1590/S0100-83582017350100015 copy

IDENTIFICATION AND VALIDATION OF REFERENCE GENES FOR THE NORMALIZATION IN REAL-TIME RT-QPCR ON RICE AND RED RICE IN COMPETITION, UNDER DIFFERENT NITROGEN DOSES

Identificação e Validação de Genes de Referência para a Normalização no Real-Time RT-qPCR em Arroz e Arroz Vermelho em Competição, sob Diferentes Doses de Nitrogênio

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

Real time reverse transcription polymerase chain reaction (RT-qPCR) is an important technique to analyze differences in gene expression due to its sensitivity, accuracy, and specificity. However, before analyzing the expression of the target gene, it is necessary to identify and evaluate the stability of candidate reference genes for the proper normalization. This study aimed at evaluating the stability of candidate reference genes in order to identify the most appropriate genes for the normalization of the transcription in rice and red rice in competition under different nitrogen levels, as well as to demonstrate the effectiveness of the reference gene selected for the expression of the cytosolic ascorbate peroxidase (OsAPX2). Eleven candidate reference genes were assessed using the RefFinder which integrates the four leading software: geNorm, NormFinder, Bestkeeper, and the comparative delta-Ct method in addition to the analysis of variance to identify genes with lower standard deviation and coefficient of variation values. Eight out of the eleven genes have shown the desired effectiveness and, among them, the gene UBC-E2 has the highest stability according to RefFinder and the analysis of variance. The expression of the gene OsAPX2 has proven to be effective in validating the candidate reference gene. This study is the first survey on the stability of candidate reference genes in rice and red rice in competition, providing information to obtain more accurate results in RT-qPCR.

Keywords:
Oryza sativa; gene expression; normalization

Sociedade Brasileira da Ciência das Plantas Daninhas Departamento de Fitotecnia - DFT, Universidade Federal de Viçosa - UFV, 36570-000 - Viçosa-MG - Brasil, Tel./Fax::(+55 31) 3899-2611 - Viçosa - MG - Brazil
E-mail: rpdaninha@gmail.com

Acompanhe os números deste periódico no seu leitor de RSS

[1] * Corresponding author: <daiane_bio@yahoo.com.br>

Gene (reference)	F (5'-3')	R (5'-3')	Efficiency rice	Efficiency red rice
ACT11 (Zhang and Hu, 2009Zhang Z., Hu J. Development and validation of endogenous reference genes for expression profiling of Medaka (Oryzias latipes) exposed to endocrine disrupting chemicals by quantitative real-time RT-PCR. Toxicol Sci. 2009;95:356-68.)	CAGCCACACTGTCCCCATCTA	AGCAAGGTCGAGACGAAGGA	2.44	2.36
β-Tubulina (Zhang and Hu, 2009Zhang Z., Hu J. Development and validation of endogenous reference genes for expression profiling of Medaka (Oryzias latipes) exposed to endocrine disrupting chemicals by quantitative real-time RT-PCR. Toxicol Sci. 2009;95:356-68.)	GCTGACCACACCTAGCTTTGG	AGGGAACCTTAGGCAGCATGT	2.87	2.83
Eef-1a (Zhang and Hu, 2007)	TTTCACTCTTGGTGTGAAGCAGAT	GACTTCCTTCACGATTTCATCGTAA	1.84	2.15
UBC-E2 (Jain et al., 2006Jain M. et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Comm. 2006;345:646-51. )	CCGTTTGTAGAGCCATAATTGCA	AGGTTGCCTGAGTCACAGTTAAGTG	2.14	2.05
eIF-4-a (Jain et al., 2006Jain M. et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Comm. 2006;345:646-51. )	TTGTGCTGGATGAAGCTGATG	GGAAGGAGCTGGAAGATATCATAGA	2.13	2.13
UBQ10 (Jain et al., 2006Jain M. et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Comm. 2006;345:646-51. )	TGGTCAGTAATCAGCCAGTTTGG	GCACCACAAATACTTGACGAACAG	1.83	1.82
UBQ5 (Jain et al., 2006Jain M. et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Comm. 2006;345:646-51. )	ACCACTTCGACCGCCACTACT	ACGCCTAAGCCTGCTGGTT	2.19	2.20
GAPDH (Jain et al., 2006Jain M. et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Comm. 2006;345:646-51. )	AAGCCAGCATCCTATGATCAGATT	CGTAACCCAGAATACCCTTGAGTTT	2.06	2.10
TIP41 - Like (Caldana et al., 2007Caldana C. et al. A quantitative RT-PCR platform for higt-throughput expression profiling of 2500 rice transcription factors. Plant Meth. 2007;3:1-9.)	GTTTGGATGAACCCCGCAA	GGCAACAAGGTCAATCCGATC	1.50	2.30
Cyclophilin (Caldana et al., 2007Caldana C. et al. A quantitative RT-PCR platform for higt-throughput expression profiling of 2500 rice transcription factors. Plant Meth. 2007;3:1-9.)	CCACCATCACAGATCGGATCTT	CGTAACCCAGAATACCCTTGAGTTT	2.17	2.03
18S rRNA (Jain et al., 2006Jain M. et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Comm. 2006;345:646-51. )	CTACGTCCCTGCCCTTTGTACA	GCAACAAGGTCAATCCGATC	2.20	2.14

		18S	Cyclophilin	Eef-1a	EIf-4a	GAPDH	UBC-E2	UBQ5	UBQ10
X	Rice	18.08	29.92	32.01	31.30	27.41	28.79	27.00	32.92
X	Red rice	18.61	30.00	29.94	30.33	26.24	28.63	26.90	24.52
CV	Rice	10.40	4.49	11.69	3.45	8.99	3.29	5.44	4.14
CV	Red rice	9.41	2.63	2.51	2.18	2.96	1.34	1.57	3.11
SD	Rice	1.88	1.34	3.74	1.08	2.46	0.94	1.35	1.03
Red rice	1.75	0.78	0.75	0.60	0.77	0.38	0.42	0.76