Optimizing stem-loop qPCR assays through multiplexed cDNA synthesis of U6 and miRNAs

Turner, Marie; Adhikari, Sajag; Subramanian, Senthil

doi:10.4161/psb.24918

Cited by 44 publications

(31 citation statements)

References 18 publications

(16 reference statements)

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“…A stem‐loop primer‐based quantitative reverse transcription PCR (SL‐qPCR) approach was used to quantify the abundance of six miRNAs in the mutant and wild‐type backgrounds (Turner et al . ). Encouragingly, minor perturbations to the abundance of three of the six quantified miRNAs, miR156, miR163 and miR168, were detected in GmDrb2ab leaves (Figure S12b).…”

Section: Resultsmentioning

confidence: 97%

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Curtin

Xiong

Michno

et al. 2017

Plant Biotechnology Journal

143

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SummaryProcessing of double‐stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL‐effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi‐allelic double mutant for the two soya bean paralogous Double‐stranded RNA‐binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9‐generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ‐line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer‐like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer‐like3 gene and the GmHen1a gene was observed in the T0 generation, but these mutations failed to transmit to the T1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole‐genome sequencing to reveal a spectrum of non‐germ‐line‐targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.

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Section: Resultsmentioning

confidence: 97%

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Curtin

Xiong

Michno

et al. 2017

Plant Biotechnology Journal

143

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“…The used primer melting temperatures (Tms) are described in Table . As internal reference we used three different nucleolar small RNAs (U1, U4 and U6) which are stable in the tested conditions according to NormFinder and BestKeeper algorithms (Andersen et al , ; Pfaffl et al , ; Turner et al , ). qPCR for DCL has been described previously (Katsarou et al , ).…”

Section: Methodsmentioning

confidence: 99%

DCL‐suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology

Κατσαρού

Mitta

Bardani

et al. 2018

Molecular Plant Pathology

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Summary RNA silencing is a universal mechanism involved in development, epigenetic modifications and responses to biotic and abiotic stresses. The major components of this mechanism are Dicer‐like (DCL), Argonaute (AGO) and RNA‐dependent RNA polymerase (RDR) proteins. Understanding the role of each component is of great scientific and agronomic importance. Plants, including Nicotiana benthamiana , an important plant model, usually possess four DCL proteins, each of which has a specific role, namely being responsible for the production of an exclusive small RNA population. Here, we used RNA interference (RNAi) technology to target DCL proteins and produced single and combinatorial mutants for DCL. We analysed the phenotype for each DCL knockdown plant, together with the small RNA profile, by next‐generation sequencing (NGS). We also investigated transgene expression, as well as viral infections, and were able to show that DCL suppression results in distinct developmental defects, changes in small RNA populations, increases in transgene expression and, finally, higher susceptibility in certain RNA viruses. Therefore, these plants are excellent tools for the following: (i) to study the role of DCL enzymes; (ii) to overexpress proteins of interest; and (iii) to understand the complex relationship between the plant silencing mechanism and biotic or abiotic stresses.

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“…Reaction conditions were as follows: 95 °C for 10 min, 95 °C for 10 s, 60 °C for 20 s and 72 °C for 10 s, for 40 cycles. Using snRNA U6 as an internal reference gene (Turner et al 2013), a melting curve analysis was performed, and the 2 −ΔΔCt method was used to analyze the data. The primers used for miRNA analysis are listed in Supplemental Table S4.…”

Section: Mirna Assaysmentioning

confidence: 99%

Dual functions of GmTOE4a in the regulation of photoperiod-mediated flowering and plant morphology in soybean

et al. 2015

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Flowering time, maturity, and plant morphology have considerable effects on the adaptation and grain yield of soybean (Glycine max). The identification of novel genes and an understanding of their molecular basis are critical to improve soybean productivity. In this study, we cloned a flowering time related APETALA2-like gene GmTOE4a and generated GmTOE4a-overexpressing lines in the cultivar Williams 82. The transgenic lines exhibited late flowering both under long day and short day conditions, and repressed the flowering-related genes, including GmFT2a, GmFT5a, GmAP1, and GmLFY, whereas the flowering repressors GmFT4 and miR156 were upregulated. Interestingly, GmTOE4a was also mediated by photoperiod via maturity genes E3 and E4, which encode photoreceptors in soybean. Further, miR172-mediated GmTOE4a, which regulates flowering in soybean, is different in Arabidopsis in that it is dependent on the CONSTANS-like gene GmCOL1a. In addition to its effect on flowering time, GmTOE4a regulated plant morphology, increased stem thickness, and reduced plant height, internode length and leaf size, which are important agronomic traits that enhance the capacity to resist lodging and increase soybean yield. This is useful information to understand the molecular mechanism of flowering time and plant morphology in soybean and will greatly influence soybean yield improvement.

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Optimizing stem-loop qPCR assays through multiplexed cDNA synthesis of U6 and miRNAs

Cited by 44 publications

References 18 publications

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

DCL‐suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology

Dual functions of GmTOE4a in the regulation of photoperiod-mediated flowering and plant morphology in soybean

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