Selection and validation of reference genes for measuring gene expression in Piper species at different life stages using RT-qPCR analysis

2021 ◽  
Author(s):  
Leandro Francisco de Oliveira ◽  
Amanda Rusiska Piovezani ◽  
Dimitre A. Ivanov ◽  
Leonardo Yoshida ◽  
Eny Iochevet Segal Floh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Life Stages ◽  
Qpcr Analysis

scholarly journals Expression Stability of Internal Reference Gene in Response to Trichoderma polysporum Infection in Avena fatua L.

2021 ◽  
Author(s):  
Haixia Zhu ◽  
Yongqiang Ma ◽  
Liang Cheng
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Avena Fatua ◽  
Expression Stability ◽  
Internal Reference ◽  
Qpcr Analysis ◽  
Suitable Combination

Abstract In order to construct a RT-qPCR system suitable for response of Avena fatua L. to Trichoderma polysporum , and screen stable internal reference genes, GeNorm, NormFinder, BestKeeper and RefFinde were used to perform SYBR Green-based RT-qPCR analysis on 8 candidate internal reference genes ( 18S , 28S , TUA , UBC , ACT , GAPDH , TBP and EF-1 ) in A. fatua samples after inoculation of T. polysporum Strain HZ-31. The results showed that TBP , 18S and UBC were the most stable internal reference genes, TBP and TUA , TBP and GAPDH , 18S and TBP , UBC and 18S were the most suitable combination of the two internal reference genes, which could be used as the internal reference genes for functional gene expression analysis during the interaction between T. polysporum and A. fatua .

scholarly journals Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Hao Xie ◽  
Bo Li ◽  
Yu Chang ◽  
Xiaoyan Hou ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Reference Genes ◽  
18S Rrna ◽  
Spinacia Oleracea ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Housekeeping Genes ◽  
Qpcr Analysis ◽  
The Stability

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, CdCl2, NaCl, NaHCO3, and Na2CO3 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.

scholarly journals Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels

2020 ◽  
Vol 21 (23) ◽  
pp. 9195
Author(s):  
Johannes Hasler ◽  
Luan Phelipe Hatt ◽  
Martin James Stoddart ◽  
Angela Rita Armiento
Keyword(s):  
Gene Expression ◽  
Osteogenic Differentiation ◽  
Reference Genes ◽  
High Throughput Screening ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Phenotype ◽  
Research Fields ◽  
Qpcr Analysis ◽  
The Stability ◽  
Suitable Reference

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) enables the monitoring of changes in cell phenotype via the high-throughput screening of numerous genes. RT-qPCR is a fundamental approach in numerous research fields, including biomaterials, yet little attention has been given to the potential impact of 3D versus monolayer (2D) cell culture and to the requirement for a constant validation of the multiple steps of gene expression analysis. The aim of this study is to use high-quality RNA to identify the most suitable reference genes for RT-qPCR analysis during the osteogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs). BM-MSCs are cultured under osteogenic conditions for 28 days in 2D or within hyaluronic acid hydrogels (3D). RNA is subject to quality controls and is then used to identify the most stable reference genes using geNorm, NormFinder, and the ∆Cq method. The effect of the reverse transcriptase is investigated, as well as the expression of osteogenic-related markers. This study shows marked differences in the stability of reference genes between 2D (RPLP0/GAPDH) and 3D (OAZ1/PPIA) culture, suggesting that it is critical to choose appropriate reference genes for 3D osteogenic cell cultures. Thus, a thorough validation under specific experimental settings is essential to obtain meaningful gene expression results.

scholarly journals Screening Reliable Reference Genes for RT-qPCR Analysis of Gene Expression in Moringa oleifera

PLoS ONE ◽  
2016 ◽  
Vol 11 (8) ◽  
pp. e0159458 ◽  
Author(s):  
Li-Ting Deng ◽  
Yu-Ling Wu ◽  
Jun-Cheng Li ◽  
Kun-Xi OuYang ◽  
Mei-Mei Ding ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Moringa Oleifera ◽  
Qpcr Analysis

scholarly journals Selection of appropriate reference genes for RT-qPCR analysis under abiotic stress and hormone treatment in celery

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7925 ◽  
Author(s):  
Kai Feng ◽  
Jie-xia Liu ◽  
Guo-Ming Xing ◽  
Sheng Sun ◽  
Sen Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Abiotic Stress ◽  
Reference Gene ◽  
Reference Genes ◽  
Regulatory Mechanism ◽  
Biological Response ◽  
Hormone Treatment ◽  
Yield And Quality ◽  
Qpcr Analysis ◽  
Selection Of

Celery is one of the most important vegetable crop and its yield and quality is influenced by many environmental factors. Researches on gene expression not only help to unravel the molecular regulatory mechanism but also identify the key genes in the biological response. RT-qPCR is a commonly used technology to quantify the gene expression. Selecting an appropriate reference gene is an effective approach to improve the accuracy of RT-qPCR assay. To our knowledge, the evaluation of reference genes under different treatments in celery has not been reported yet. In this study, the expression stabilities of eight candidate reference genes (ACTIN, eIF-4α, GAPDH, TBP, TUB-A, UBC, TUB-B, and EF-1α) under abiotic stresses (heat, cold, drought, and salt) and hormone treatments (SA, MeJA, GA, and ABA) were detected. The expression stabilities of candidate genes were compared and ranked by geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder programs. The results calculated by different programs were not completely consistent. Considering the comprehensive analysis results, ACTIN was the most stable reference gene and TUB-B showed the worst expression stabilities under the selected abiotic stress and hormone treatments in celery. The reliability of reference genes was further confirmed by the normalization of CAT1 gene under drought stress. This study presented evidences and basis to select the appropriate reference genes under different treatments in celery.

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