Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs
TLDR
In this article, an end-point and real-time looped RT-PCR procedure was proposed to detect miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from 0.1 μl of phloem sap.Abstract:
MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.read more
Citations
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Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways
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Regulation of Flowering by Trehalose-6-Phosphate Signaling in Arabidopsis thaliana
Vanessa Wahl,Jathish Ponnu,Armin Schlereth,Stéphanie Arrivault,Tobias Langenecker,Annika Franke,Regina Feil,John E. Lunn,Mark Stitt,Markus Schmid +9 more
TL;DR: It is shown that the T6P pathway affects flowering both in the leaves and at the shoot meristem, and TPS1 is integrated into the existing genetic framework of flowering-time control.
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Transcriptional Control of Gene Expression by MicroRNAs
Basel Khraiwesh,M. Asif Arif,Gotelinde I. Seumel,Stephan Ossowski,Detlef Weigel,Ralf Reski,Wolfgang Frank +6 more
TL;DR: It is shown here that in moss Physcomitrella patens mutants without a DICER-LIKE1b gene, maturation of miRNAs is normal but cleavage of target RNAs is abolished and levels of these transcripts are drastically reduced, suggesting initiation of epigenetic silencing by DNA methylation depends on the ratio of the miRNA and its target RNA.
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Differential expression of miRNAs in response to salt stress in maize roots
TL;DR: Salt-responsive miRNAs are involved in the regulation of metabolic, morphological and physiological adaptations of maize seedlings at the post-transcriptional level and might explain the distinct salt sensitivities between maize lines.
References
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MicroRNAs: Genomics, Biogenesis, Mechanism, and Function
TL;DR: Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.
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Journal ArticleDOI
Prediction of Plant MicroRNA Targets
Matthew W. Rhoades,Brenda J. Reinhart,Lee P. Lim,Christopher B. Burge,Bonnie Bartel,David P. Bartel +5 more
TL;DR: This work predicts regulatory targets for 14 Arabidopsis microRNAs (miRNAs) by identifying mRNAs with near complementarity and identifies members of transcription factor gene families involved in developmental patterning or cell differentiation.
Journal ArticleDOI
Novel and Stress-Regulated MicroRNAs and Other Small RNAs from Arabidopsis
Ramanjulu Sunkar,Jian-Kang Zhu +1 more
TL;DR: It is suggested that a large number of miRNAs and other small regulatory RNAs are encoded by the Arabidopsis genome and that some of them may play important roles in plant responses to environmental stresses as well as in development and genome maintenance.