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1.
J Biomol Tech ; 31(2): 47-56, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-31966025

RESUMEN

Small RNAs (smRNAs) are important regulators of many biologic processes and are now most frequently characterized using Illumina sequencing. However, although standard RNA sequencing library preparation has become routine in most sequencing facilities, smRNA sequencing library preparation has historically been challenging because of high input requirements, laborious protocols involving gel purifications, inability to automate, and a lack of benchmarking standards. Additionally, studies have suggested that many of these methods are nonlinear and do not accurately reflect the amounts of smRNAs in vivo. Recently, a number of new kits have become available that permit lower input amounts and less laborious, gel-free protocol options. Several of these new kits claim to reduce RNA ligase-dependent sequence bias through novel adapter modifications and to lessen adapter-dimer contamination in the resulting libraries. With the increasing number of smRNA kits available, understanding the relative strengths of each method is crucial for appropriate experimental design. In this study, we systematically compared 9 commercially available smRNA library preparation kits as well as NanoString probe hybridization across multiple study sites. Although several of the new methodologies do reduce the amount of artificially over- and underrepresented microRNAs (miRNAs), we observed that none of the methods was able to remove all of the bias in the library preparation. Identical samples prepared with different methods show highly varied levels of different miRNAs. Even so, many methods excelled in ease of use, lower input requirement, fraction of usable reads, and reproducibility across sites. These differences may help users select the most appropriate methods for their specific question of interest.


Asunto(s)
Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/normas , MicroARNs/genética , Análisis de Secuencia de ARN/normas , MicroARNs/aislamiento & purificación , Reproducibilidad de los Resultados , Programas Informáticos
2.
Metallomics ; 9(7): 876-890, 2017 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-28620661

RESUMEN

Iron (Fe) is required for plant health, but it can also be toxic when present in excess. Therefore, Fe levels must be tightly controlled. The Arabidopsis thaliana E3 ligase BRUTUS (BTS) is involved in the negative regulation of the Fe deficiency response and we show here that the two A. thaliana BTS paralogs, BTS LIKE1 (BTSL1) and BTS LIKE2 (BTSL2) encode proteins that act redundantly as negative regulators of the Fe deficiency response. Loss of both of these E3 ligases enhances tolerance to Fe deficiency. We further generated a triple mutant with loss of both BTS paralogs and a partial loss of BTS expression that exhibits even greater tolerance to Fe-deficient conditions and increased Fe accumulation without any resulting Fe toxicity effects. Finally, we identified a mutant carrying a novel missense mutation of BTS that exhibits an Fe deficiency response in the root when grown under both Fe-deficient and Fe-sufficient conditions, leading to Fe toxicity when plants are grown under Fe-sufficient conditions.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Deficiencias de Hierro , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Genes de Plantas , Hierro/metabolismo , Hierro/toxicidad , Modelos Biológicos , Mutación/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Semillas/efectos de los fármacos , Semillas/metabolismo , Homología de Secuencia de Aminoácido , Transcriptoma/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética
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