Internal standards for quantitative RT-PCR studies of gene expression under drought treatment in barley (Hordeum vulgare L.): the effects of developmental stage and leaf age

Rapacz, Marcin; Stępień, Agata; Skorupa, K

doi:10.1007/s11738-012-0967-1

Cited by 71 publications

(54 citation statements)

References 40 publications

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“…Expression values were normalised against the ADP-ribosylation factor gene, which according to geNorm PLUS analysis (data not shown), displayed the highest stability of expression level. It was also suggested as the most suitable to study drought-induced changes in gene expression at the seedling stage in barley (Rapacz et al 2012). Expression values were calculated using the 2 −ΔΔCT method (Schmittgen and Livak 2008).…”

Section: Methodsmentioning

confidence: 99%

Genome-wide identification, characterisation and expression profiles of calcium-dependent protein kinase genes in barley (Hordeum vulgare L.)

et al. 2016

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In plant cells, calcium-dependent protein kinases (CDPKs) are important sensors of Ca2+ flux resulting from various environmental stresses like cold, drought or salt stress. Previous genome sequence analysis and comparative studies in Arabidopsis (Arabidopsis thaliana L.) and rice (Oryza sativa L.) defined a multi-gene family of CDPKs. Here, we identified and characterised the CDPK gene complement of the model plant, barley (Hordeum vulgare L.). Comparative analysis encompassed phylogeny reconstruction based on newly available barley genome sequence, as well as established model genomes (e.g. O. sativa, A. thaliana, Brachypodium distachyon). Functional gene copies possessed characteristic CDPK domain architecture, including a serine/threonine kinase domain and four regulatory EF-hand motifs. In silico verification was followed by measurements of transcript abundance via real-time polymerase chain reaction (PCR). The relative expression of CDPK genes was determined in the vegetative growth stage under intensifying drought stress conditions. The majority of barley CDPK genes showed distinct changes in patterns of expression during exposure to stress. Our study constitutes evidence for involvement of the barley CDPK gene complement in signal transduction pathways relating to adaptation to drought. Our bioinformatics and transcriptomic analyses will provide an important foundation for further functional dissection of the barley CDPK gene family.Electronic supplementary materialThe online version of this article (doi:10.1007/s13353-016-0357-2) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Genome-wide identification, characterisation and expression profiles of calcium-dependent protein kinase genes in barley (Hordeum vulgare L.)

et al. 2016

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“…Each real-time PCR reaction was performed independently for three biological replicates, and for every biological replicate three (splicing isoforms analysis) or two (pri-miRNA abundance analysis) technical replicates were performed. The barley ADP-ribosylation factor 1-like [GenBank: AJ508228.2] gene fragment of 61 nt was simultaneously amplified and detected as an internal reference [77]. Expression levels were calculated with the relative quantification method (2 -ΔCt ) as fold-change value and presented in a form of log 10 2 -ΔCt .…”

Section: Methodsmentioning

confidence: 99%

Developmentally regulated expression and complex processing of barley pri-microRNAs

et al. 2013

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BackgroundMicroRNAs (miRNAs) regulate gene expression via mRNA cleavage or translation inhibition. In spite of barley being a cereal of great economic importance, very little data is available concerning its miRNA biogenesis. There are 69 barley miRNA and 67 pre-miRNA sequences available in the miRBase (release 19). However, no barley pri-miRNA and MIR gene structures have been shown experimentally. In the present paper, we examine the biogenesis of selected barley miRNAs and the developmental regulation of their pri-miRNA processing to learn more about miRNA maturation in barely.ResultsTo investigate the organization of barley microRNA genes, nine microRNAs - 156g, 159b, 166n, 168a-5p/168a-3p, 171e, 397b-3p, 1120, and 1126 - were selected. Two of the studied miRNAs originate from one MIR168a-5p/168a-3p gene. The presence of all miRNAs was confirmed using a Northern blot approach. The miRNAs are encoded by genes with diverse organizations, representing mostly independent transcription units with or without introns. The intron-containing miRNA transcripts undergo complex splicing events to generate various spliced isoforms. We identified miRNAs that were encoded within introns of the noncoding genes MIR156g and MIR1126. Interestingly, the intron that encodes miR156g is spliced less efficiently than the intron encoding miR1126 from their specific precursors. miR397b-3p was detected in barley as a most probable functional miRNA, in contrast to rice where it has been identified as a complementary partner miRNA*. In the case of miR168a-5p/168a-3p, we found the generation of stable, mature molecules from both pre-miRNA arms, confirming evolutionary conservation of the stability of both species, as shown in rice and maize. We suggest that miR1120, located within the 3′ UTR of a protein-coding gene and described as a functional miRNA in wheat, may represent a siRNA generated from a mariner-like transposable element.ConclusionsSeven of the eight barley miRNA genes characterized in this study contain introns with their respective transcripts undergoing developmentally specific processing events prior to the dicing out of pre-miRNA species from their pri-miRNA precursors. The observed tendency to maintain the intron encoding miR156g within the transcript, and preferences in splicing the miR1126-harboring intron, may suggest the existence of specific regulation of the levels of intron-derived miRNAs in barley.

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“…Total RNA was quantified using the Experion TM RNA StdSens microcapillary chip (Bio-Rad, Mississauga, ON, Canada) and its integrity based on the RNA quality indicator (RQI) calculated by the Experion TM software was always greater than 8. First-strand cDNA was synthesized from 1 µg of total RNA and oligo(dT) 18 primers using the Transcriptor First Strand cDNA synthesis Kit (Roche Applied Science, Laval, QC, Canada) following the manufacturer instructions. Any residual genomic DNA was removed by a treatment with DNaseI (Invitrogen, Burlington, ON, Canada) prior to cDNA synthesis.…”

Section: Rna Extraction and Cdna Synthesismentioning

confidence: 99%

“…Act has been previously shown to be unstable under various experimental conditions and was deemed unsuitable to normalize stress-induced gene expression in several species [12] [21] [32] including alfalfa [30]. Rapacz et al [18] concluded that the stability of Act in barley (Hordeum vulgare) exposed to abiotic stress could depend on the developmental stage. Tub is another gene commonly used for normalization of gene expression that has been frequently shown to be unstable across species and experimental treatments [9] [12] [16] [20] [30].…”

Section: Stability Of the Expression Of Candidate Reference Genesmentioning

confidence: 99%

Reference Genes for RT-qPCR Analysis of Environmentally and Developmentally Regulated Gene Expression in Alfalfa

Castonguay¹,

Jl²,

Dubé³

2015

AJPS

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Reverse transcription quantitative PCR (RT-qPCR) is a highly sensitive technique that has become the standard for the analysis of differences in gene expression in response to experimental treatments or among genetic sources. The accuracy of the RT-qPCR results can be significantly affected by uncontrolled sources of variation that can be accounted for normalization with so-called reference genes stably expressed under various conditions. In this study we assessed the stability of 21 reference gene candidates in crowns of two alfalfa cultivars (Apica and Evolution) exposed to various environmental conditions (cold, water stress and photoperiod) and from above ground biomass of the cultivar Orca sampled at three developmental stages (vegetative, full bloom and mature pods). Candidates were selected based on their previous identification in other plant species or their stable expression in a differential hybridization of alfalfa ESTs with cDNA from nonacclimated and cold-acclimated alfalfa. Genes encoding ubiquitin protein ligase 2a (UBL-2a), actin depolymerizing factor (ADF) and retention in endoplasmic reticulum 1 protein (Rer1) were the most stable across experimental conditions. Conversely β-actin (Act), α-tubulin (Tub) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) frequently used as "housekeeping genes" in gene expression studies showed poor stability. No more than two reference genes were required to normalize the gene expression data under each condition. Normalization of the expression of genes of interest with unstable reference genes led to observations that were conflicting with those made with validated reference genes and that were in some cases inconsistent with the current knowledge of the trait. The reference genes identified in this study are strong candidates for normalization of gene expression in cultivated alfalfa.

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Internal standards for quantitative RT-PCR studies of gene expression under drought treatment in barley (Hordeum vulgare L.): the effects of developmental stage and leaf age

Cited by 71 publications

References 40 publications

Genome-wide identification, characterisation and expression profiles of calcium-dependent protein kinase genes in barley (Hordeum vulgare L.)

Genome-wide identification, characterisation and expression profiles of calcium-dependent protein kinase genes in barley (Hordeum vulgare L.)

Developmentally regulated expression and complex processing of barley pri-microRNAs

Reference Genes for RT-qPCR Analysis of Environmentally and Developmentally Regulated Gene Expression in Alfalfa

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