Reference Gene Selection for Gene Expression Analyses in Mouse Models of Acute Lung Injury

qRT-PCR still remains the most widely used method for quantifying gene expression levels, although newer technologies such as next generation sequencing are becoming increasingly popular. A critical, yet often underappreciated, problem when analysing qRT-PCR data is the selection of suitable reference genes. This problem is compounded in situations where up to 25% of all genes may change (e.g., due to leukocyte invasion), as is typically the case in ARDS. Here, we examined 11 widely used reference genes for their suitability in commonly used models of acute lung injury (ALI): ventilator-induced lung injury (VILI), in vivo and ex vivo, lipopolysaccharide plus mechanical ventilation (MV), and hydrochloric acid plus MV. The stability of reference gene expression was determined using the NormFinder, BestKeeper, and geNorm algorithms. We then proceeded with the geNorm results because this is the only algorithm that provides the number of reference genes required to achieve normalisation. We chose interleukin-6 (Il‑6) and C-X-C motif ligand 1 (Cxcl-1) as the genes of interest to analyse and demonstrate the impact of inappropriate normalisation. Reference gene stability differed between the ALI models and even within the subgroup of VILI models, no common reference gene index (RGI) could be determined. NormFinder, BestKeeper, and geNorm produced slightly different, but comparable results. Inappropriate normalisation of Il-6 and Cxcl1 gene expression resulted in significant misinterpretation in all four ALI settings. In conclusion, choosing an inappropriate normalisation strategy can introduce different kinds of bias such as gain or loss as well as under- or overestimation of effects, affecting the interpretation of gene expression data.

Download Full-text

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

Scientific Reports ◽

10.1038/s41598-021-81524-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Min-dong Chen ◽

Bin Wang ◽

Yong-ping Li ◽

Mei-juan Zeng ◽

Jian-ting Liu ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Expression Patterns ◽

Suitable Reference Gene ◽

Expression Levels ◽

Qrt Pcr ◽

Suitable Reference ◽

Gene Expression Levels

AbstractSelecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H2O2, and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.

Download Full-text

Identification of Reference Genes for Quantitative Gene Expression Studies in Pinus massoniana and Its Introgression Hybrid

Forests ◽

10.3390/f10090787 ◽

2019 ◽

Vol 10 (9) ◽

pp. 787

Author(s):

Jiaxing Mo ◽

Jin Xu ◽

Wenjing Jin ◽

Liwei Yang ◽

Tongming Yin ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Regulation Of Gene Expression ◽

Threshold Value ◽

Pinus Massoniana ◽

Tissue Type ◽

Amplification Efficiency ◽

Qrt Pcr

qRT-PCR is a powerful molecular research tool to study the regulation of gene expression. However, to accurately calculate gene expression levels, an experiment should include proper reference genes that show no changes in their expression level. Pinus massoniana, P. hwangshanensis, and their introgression hybrid in Mountain Lushan, China, are an ideal model for studying introgression and speciation. Although some research on reference gene selection for P. massoniana has been reported before, no studies on this subject have been performed where P. massoniana and its introgression hybrid were evaluated simultaneously. Here, we investigated ten genes (upLOC, SDH, ACT, EF, TOC75, DMWD, FBOX, PGK1, UBQ, and CL2417C7) identified from transcriptome data of these two taxa for reference gene potential. These ten genes were then screened across multiple tissues such as cone, young and mature stems, and young needles according to qRT-PCR thermal cycling and dissociation. Correlation coefficient, amplification efficiency, and cycle threshold value (Ct) range were applied to evaluate the reliability of each gene. The stability of candidate reference gene expression was calculated using three algorithms: geNorm, NormFinder, and BestKeeper. Base on the reliability and stability, we then offered a list of genes of recommended and not recommended for seven different tissue type and species. Our results demonstrated that different sample lines require different genes as reference to evaluate.

Download Full-text

Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽

10.3390/genes12070960 ◽

2021 ◽

Vol 12 (7) ◽

pp. 960

Author(s):

Meagan Archer ◽

Jianping Xu

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Quantitative Polymerase Chain Reaction ◽

Specific Method ◽

Experimental Conditions ◽

Reference Gene Selection ◽

Physiological Processes ◽

The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

Download Full-text

Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

Scientific Reports ◽

10.1038/s41598-021-92780-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Madhab Kumar Sen ◽

Kateřina Hamouzová ◽

Pavlina Košnarová ◽

Amit Roy ◽

Josef Soukup

Keyword(s):

Reference Gene ◽

Herbicide Resistance ◽

Reference Genes ◽

Gene Selection ◽

High Yield ◽

Suitable Reference Gene ◽

Grass Species ◽

Experimental Conditions ◽

Qrt Pcr ◽

Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

Download Full-text

Identification and validation of miRNA reference genes in poplar under pathogen stress

10.21203/rs.3.rs-225734/v1 ◽

2021 ◽

Author(s):

Lichun Zhang ◽

Xiaoqian Yang ◽

Yiyi Yin ◽

Jinxing Wang ◽

Yanwei Wang

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Small Rna ◽

Reference Genes ◽

Expression Profiles ◽

Small Rna Sequencing ◽

Rna Seq ◽

Internal Standards ◽

Qrt Pcr ◽

Canker Pathogen

Abstract Quantitative real time polymerase chain reaction (qRT-PCR) is a common method to analyze gene expression. Due to differences in RNA quantity, quality, and reverse transcription efficiency between qRT-PCR samples, reference genes are used as internal standards to normalize gene expression. However, few universal genes especially miRNAs have been identified as reference so far. Therefore, it is essential to identify reference genes that can be used across various experimental conditions, stress treatments, or tissues. In this study, 14 microRNAs (miRNAs) and 5.8S rRNA were assessed for expression stability in poplar trees infected with canker pathogen. Using three reference gene analysis programs, we found that miR156g and miR156a exhibited stable expression throughout the infection process. miR156g and miR156a were then tested as internal standards to measure the expression of miR1447 and miR171c, and the results were compared to small RNA sequencing (RNA-seq) data. We found that when miR156a was used as the reference gene, the expression of miR1447 and miR171c were consistent with the small RNA-seq expression profiles. Therefore, miR156a was the most stable miRNAs examined in this study, and could be used as a reference gene in poplar under canker pathogen stress, which should enable comprehensive comparisons of miRNAs expression and avoid the bias caused by different lenth between detected miRNAs and traditional referece genes. The present study has expanded the miRNA reference genes available for gene expression studies in trees under biotic stress.

Download Full-text

Metformin-stimulated AMPK-α1 promotes microvascular repair in acute lung injury

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00173.2013 ◽

2013 ◽

Vol 305 (11) ◽

pp. L844-L855 ◽

Cited By ~ 47

Author(s):

Ming-Yuan Jian ◽

Mikhail F. Alexeyev ◽

Paul E. Wolkowicz ◽

Jaroslaw W. Zmijewski ◽

Judy R. Creighton

Keyword(s):

Endothelial Cells ◽

Acute Lung Injury ◽

Lung Injury ◽

Ex Vivo ◽

Endothelial Permeability ◽

Beneficial Effects ◽

Isolated Lung

Acute lung injury secondary to sepsis is a leading cause of mortality in sepsis-related death. Present therapies are not effective in reversing endothelial cell dysfunction, which plays a key role in increased vascular permeability and compromised lung function. AMP-activated protein kinase (AMPK) is a molecular sensor important for detection and mediation of cellular adaptations to vascular disruptive stimuli. In this study, we sought to determine the role of AMPK in resolving increased endothelial permeability in the sepsis-injured lung. AMPK function was determined in vivo using a rat model of endotoxin-induced lung injury, ex vivo using the isolated lung, and in vitro using cultured rat pulmonary microvascular endothelial cells (PMVECs). AMPK stimulation using N1-(α-d-ribofuranosyl)-5-aminoimidizole-4-carboxamide or metformin decreased the LPS-induced increase in permeability, as determined by filtration coefficient ( Kf) measurements, and resolved edema as indicated by decreased wet-to-dry ratios. The role of AMPK in the endothelial response to LPS was determined by shRNA designed to decrease expression of the AMPK-α1 isoform in capillary endothelial cells. Permeability, wounding, and barrier resistance assays using PMVECs identified AMPK-α1 as the molecule responsible for the beneficial effects of AMPK in the lung. Our findings provide novel evidence for AMPK-α1 as a vascular repair mechanism important in the pulmonary response to sepsis and identify a role for metformin treatment in the management of capillary injury.

Download Full-text

Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis)

Bulletin of Entomological Research ◽

10.1017/s0007485316000948 ◽

2016 ◽

Vol 107 (3) ◽

pp. 359-368 ◽

Cited By ~ 12

Author(s):

Y. Tan ◽

X.-R. Zhou ◽

B.-P. Pang

Keyword(s):

Reference Gene ◽

Reference Genes ◽

Target Genes ◽

Gene Selection ◽

Developmental Stages ◽

Pcr Analysis ◽

Experimental Conditions ◽

Reference Gene Selection ◽

Functional Studies ◽

Qrt Pcr

AbstractQuantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔCt method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.

Download Full-text

Reference Gene Selection for miRNA qRT-PCR Analysis in Lily

10.21203/rs.3.rs-102523/v1 ◽

2020 ◽

Author(s):

Qian Zhang ◽

Xue Gao ◽

Lian-Juan Wang ◽

Yu-Qian Zhao ◽

Gui-Xia Jia

Keyword(s):

Reference Gene ◽

Stress Responses ◽

Reference Genes ◽

Gene Selection ◽

Developmental Stages ◽

Pcr Analysis ◽

Critical Element ◽

Biotic And Abiotic Stress ◽

Experimental Conditions ◽

Qrt Pcr

Abstract Background: The selection of reliable reference genes is a critical element for obtaining accurate gene expression data to assess quantitative real-time polymerase chain reaction (qRT-PCR) performance. It is critical to use suitable reference genes in miRNA qRT-PCR because of short amplification products and large differences in the expression levels of target miRNAs involved in some biological processes. However, in lily, which exhibits a large complex genome but lacks a reference, the available miRNA reference genes for use in qRT-PCR under various treatment conditions are limited, and their reliability has rarely been systematically evaluated.Results: In this study, 8 candidate reference genes, including three classic housekeeping genes and five potential miRNAs from the miRNA library of L. × formolongi, were selected and assessed for expression stability utilizing the BestKeeper, geNorm and Normfinder tools, together with the Delta Ct method, across a diverse set of biotic and abiotic experimental conditions (developmental stages, tissues, heat stress and pathogen defence) to determine the best reference gene(s) for L. × formolongi and L. regale. The final ranking was reordered by using RankAggreg, and the results showed that the novel miRNA PC-3p-67_108977 and the conserved miRNAs miR399a, miR399a and U6 were the most stable genes for L. × formolongi and L. regale, respectively, under all tested experimental conditions. Additionally, PC-3p-67_108977 and U6 were the most suitable genes for qRT-PCR studies in lily.Conclusions: This study provides a comprehensive evaluation of the reliability of reference genes for miRNA studies on development and biotic and abiotic stress responses in different lilies. These results will be beneficial for miRNA identification and functional studies of lilies in the future.

Download Full-text

Validation of Reference Genes for Quantitative Real-time PCR in Diaphanosoma Celebensis: For Chemical Exposure Conditions and Different Ages

10.21203/rs.3.rs-847536/v1 ◽

2021 ◽

Author(s):

Young-Mi Lee ◽

Soyeon In ◽

Se-Joo Kim ◽

Eun-Ji Won ◽

Hayoung Cho ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Expression Profiles ◽

Chemical Exposure ◽

Mrna Levels ◽

Qrt Pcr ◽

Different Ages ◽

Diaphanosoma Celebensis

Abstract Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a primary approach for evaluating gene expression, requires an appropriate normalization strategy to rule out variations in gene expression among samples. The best option is to use a reference gene whose expression level is stable across various experimental conditions to compare the mRNA levels of a target gene. However, there is limited information on how the reference gene is differentially expressed at different ages (growth) in small invertebrates with notable changes such as molting. In this study, expression profiles of nine candidate reference genes from the brackish water flea, Diaphanosoma celebensis, were evaluated under diverse exposure to toxicants and according to growth. As a result, four different algorithms showed similar stabilities of genes for chemical exposures in the case of limited conditions using the same developmental stage (e.g., adult), while the results according to age showed a significantly different pattern in suite of candidate reference genes. This affected the results of genes EcRA and GST, which are involved in development and detoxification mechanisms, respectively. Our finding is the first step towards establishing a standardized real-time qRT-PCR analysis of this environmentally important invertebrate that has potential for aquatic ecotoxicology, particularly in estuarine environments.

Download Full-text

Identification of suitable reference genes of Scylla paramamosain for gene expression profiling in various tissues and under vibrio challenge

Crustaceana ◽

10.1163/15685403-00003823 ◽

2018 ◽

Vol 91 (10) ◽

pp. 1195-1210 ◽

Cited By ~ 2

Author(s):

Yabo Fang ◽

Le Diao ◽

Fengying Zhang ◽

Lingbo Ma ◽

Mengdi Zhao ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

18S Rrna ◽

Elongation Factor ◽

Scylla Paramamosain ◽

Mud Crab ◽

Expression Levels ◽

Qrt Pcr ◽

Elongation Factor 1

Abstract The quantitative real-time transcription-polymerase chain reaction (qRT-PCR) is now used widely in studies about mRNA expression levels. The selection of one or more stable reference gene(s) used for data normalization is substantial. In this study, expression levels of eleven candidate reference genes (β-actin, 16S rRNA, 18S rRNA, 28S rRNA, α-I tubulin, GAPDH, ribosomal protein L13, elongation factor 1 α, elongation factor 2, arginine kinase and ubiquitin) were examined using the GenomeLab GeXP analysis system (Beckman Coulter). Gene expression data were analysed using two different statistical models: geNorm and NormFinder. (1) In six different tissues (hepatopancreas, haemocytes, heart, gill, muscle, and testis) from the mud crab, Scylla paramamosain, 18S rRNA and elongation factor 1 α were identified as the two best reference genes. (2) In the haemocytes after being challenged by Vibro parahaemolyticus, the result suggested that ubiquitin was the most stable gene after the treatment. 18S rRNA, elongation factor 1 α and ubiquitin are herein recommended as the best combination. These results provide useful options for reference gene selection under different experimental conditions in qRT-PCR studies in the mud crab.

Download Full-text