Identification of reference genes for quantitative expression analysis of microRNAs and mRNAs in barley under various stress conditions.
Identification of reference genes for quantitative expression analysis of microRNAs and mRNAs in barley under various stress conditions.
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DOI：
10.1371/journal.pone.0118503
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Tricker PJ
中科院分区：
文献类型：
--
作者：
Ferdous J;Li Y;Reid N;Langridge P;Shi BJ;Tricker PJ
为了使用实时定量逆转录聚合酶链式反应(QPCR)进行准确可靠的基因表达分析，选择合适的参考基因作为内对照进行标准化是至关重要的。我们推测，非编码的小核仁RNA(SnoRNAs)在不同的大麦品种和不同的实验处理下，在不同的组织和植物生长的不同发育阶段都有稳定的表达，因此可能被证明是适合于microRNAs(MiRNAs)和mRNAs表达分析的参考基因。在本研究中，我们研究了6个大麦品种的10个候选参考基因在干旱、真菌侵染、硼毒害、营养缺乏和盐分五种胁迫下的表达稳定性。我们比较了四个常用的看家基因：肌动蛋白(Act)、α-微管蛋白(α-TUB)、糖酵解甘油醛-3-磷酸脱氢酶(GAPDH)、腺苷二磷酸核糖化因子1样蛋白(ADP)、四个snoRNAs(U18、U61、snoR14和snoR23)和两个microRNAs(MIR168、miR159)。我们发现，在不同的样本中，ADP、snoR14和snoR23是这些候选基因中最好的。为了使用实时定量逆转录聚合酶链式反应(QPCR)进行准确可靠的基因表达分析，选择合适的参考基因作为内对照进行标准化是至关重要的。我们推测，非编码的小核仁RNA(SnoRNAs)在不同的大麦品种和不同的实验处理下，在不同的组织和植物生长的不同发育阶段都有稳定的表达，因此可能被证明是适合于microRNAs(MiRNAs)和mRNAs表达分析的参考基因。在本研究中，我们研究了6个大麦品种的10个候选参考基因在干旱、真菌侵染、硼毒害、营养缺乏和盐分五种胁迫下的表达稳定性。我们比较了四个常用的看家基因：肌动蛋白(Act)、α-微管蛋白(α-TUB)、糖酵解甘油醛-3-磷酸脱氢酶(GAPDH)、腺苷二磷酸核糖化因子1样蛋白(ADP)、四个snoRNAs(U18、U61、snoR14和snoR23)和两个microRNAs(MIR168、miR159)。我们发现，在不同的样本中，ADP、snoR14和snoR23是这些候选基因中最好的。此外，我们还发现MIR168基因可以作为大麦基因表达分析的参考基因。最后，我们验证了我们的稳定和不稳定候选参考基因在不同胁迫条件下对mRNA和miRNA qPCR数据进行归一化的性能，并展示了稳定候选参考基因的优越性。我们的数据证明了大麦snoRNAs和miRNAs在不同胁迫处理下作为miRNA和mRNAqPCR数据归一化的潜在参考基因的适用性。
For accurate and reliable gene expression analysis using quantitative real-time reverse transcription PCR (qPCR), the selection of appropriate reference genes as an internal control for normalization is crucial. We hypothesized that non-coding, small nucleolar RNAs (snoRNAs) would be stably expressed in different barley varieties and under different experimental treatments, in different tissues and at different developmental stages of plant growth and therefore might prove to be suitable reference genes for expression analysis of both microRNAs (miRNAs) and mRNAs. In this study, we examined the expression stability of ten candidate reference genes in six barley genotypes under five experimental stresses, drought, fungal infection, boron toxicity, nutrient deficiency and salinity. We compared four commonly used housekeeping genes; Actin (ACT), alpha-Tubulin (α-TUB), Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ADP-ribosylation factor 1-like protein (ADP), four snoRNAs; (U18, U61, snoR14 and snoR23) and two microRNAs (miR168, miR159) as candidate reference genes. We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples. For accurate and reliable gene expression analysis using quantitative real-time reverse transcription PCR (qPCR), the selection of appropriate reference genes as an internal control for normalization is crucial. We hypothesized that non-coding, small nucleolar RNAs (snoRNAs) would be stably expressed in different barley varieties and under different experimental treatments, in different tissues and at different developmental stages of plant growth and therefore might prove to be suitable reference genes for expression analysis of both microRNAs (miRNAs) and mRNAs. In this study, we examined the expression stability of ten candidate reference genes in six barley genotypes under five experimental stresses, drought, fungal infection, boron toxicity, nutrient deficiency and salinity. We compared four commonly used housekeeping genes; Actin (ACT), alpha-Tubulin (α-TUB), Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ADP-ribosylation factor 1-like protein (ADP), four snoRNAs; (U18, U61, snoR14 and snoR23) and two microRNAs (miR168, miR159) as candidate reference genes. We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples. Additionally, we found that miR168 was a suitable reference gene for expression analysis in barley. Finally, we validated the performance of our stable and unstable candidate reference genes for both mRNA and miRNA qPCR data normalization under different stress conditions and demonstrated the superiority of the stable candidates. Our data demonstrate the suitability of barley snoRNAs and miRNAs as potential reference genes for miRNA and mRNA qPCR data normalization under different stress treatments.
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影响因子：
14.9
作者：
Chen C;Ridzon DA;Broomer AJ;Zhou Z;Lee DH;Nguyen JT;Barbisin M;Xu NL;Mahuvakar VR;Andersen MR;Lao KQ;Livak KJ;Guegler KJ
通讯作者：
Guegler KJ
影响因子：
2.9
作者：
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通讯作者：
Margis, Rogerio
影响因子：
2.7
作者：
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通讯作者：
Zumla, A
影响因子：
3.7
作者：
Carnavale Bottino M;Rosario S;Grativol C;Thiebaut F;Rojas CA;Farrineli L;Hemerly AS;Ferreira PC
通讯作者：
Ferreira PC
影响因子：
4.6
作者：
Brattelid, Trond;Aarnes, Eva-Katrine;Jonassen, Anne K.
通讯作者：
Jonassen, Anne K.