Optimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.

There are several protocols and kits for the extraction of circulating RNAs from plasma with a following quantification of specific genes via RT-qPCR. Due to the marginal amount of cell-free RNA in plasma samples, the total RNA yield is insufficient to perform Next-Generation Sequencing (NGS), the s...

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Autores principales:	Melanie Spornraft, Benedikt Kirchner, Bettina Haase, Vladimir Benes, Michael W Pfaffl, Irmgard Riedmaier
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5272204a5ba445689dc74329bdfed01a
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spelling	oai:doaj.org-article:5272204a5ba445689dc74329bdfed01a2021-11-25T06:00:17ZOptimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.1932-620310.1371/journal.pone.0107259https://doaj.org/article/5272204a5ba445689dc74329bdfed01a2014-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0107259https://doaj.org/toc/1932-6203There are several protocols and kits for the extraction of circulating RNAs from plasma with a following quantification of specific genes via RT-qPCR. Due to the marginal amount of cell-free RNA in plasma samples, the total RNA yield is insufficient to perform Next-Generation Sequencing (NGS), the state-of-the-art technology in massive parallel sequencing that enables a comprehensive characterization of the whole transcriptome. Screening the transcriptome for biomarker signatures accelerates progress in biomarker profiling for molecular diagnostics, early disease detection or food safety. Therefore, the aim was to optimize a method that enables the extraction of sufficient amounts of total RNA from bovine plasma to generate good-quality small RNA Sequencing (small RNA-Seq) data. An increased volume of plasma (9 ml) was processed using the Qiagen miRNeasy Serum/Plasma Kit in combination with the QIAvac24 Plus system, a vacuum manifold that enables handling of high volumes during RNA isolation. 35 ng of total RNA were passed on to cDNA library preparation followed by small RNA high-throughput sequencing analysis on the Illumina HiSeq2000 platform. Raw sequencing reads were processed by a data analysis pipeline using different free software solutions. Seq-data was trimmed, quality checked, gradually selected for miRNAs/piRNAs and aligned to small RNA reference annotation indexes. Mapping to human reference indexes resulted in 4.8±2.8% of mature miRNAs and 1.4±0.8% of piRNAs and of 5.0±2.9% of mature miRNAs for bos taurus.Melanie SpornraftBenedikt KirchnerBettina HaaseVladimir BenesMichael W PfafflIrmgard RiedmaierPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 9, p e107259 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Melanie Spornraft Benedikt Kirchner Bettina Haase Vladimir Benes Michael W Pfaffl Irmgard Riedmaier Optimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.
description	There are several protocols and kits for the extraction of circulating RNAs from plasma with a following quantification of specific genes via RT-qPCR. Due to the marginal amount of cell-free RNA in plasma samples, the total RNA yield is insufficient to perform Next-Generation Sequencing (NGS), the state-of-the-art technology in massive parallel sequencing that enables a comprehensive characterization of the whole transcriptome. Screening the transcriptome for biomarker signatures accelerates progress in biomarker profiling for molecular diagnostics, early disease detection or food safety. Therefore, the aim was to optimize a method that enables the extraction of sufficient amounts of total RNA from bovine plasma to generate good-quality small RNA Sequencing (small RNA-Seq) data. An increased volume of plasma (9 ml) was processed using the Qiagen miRNeasy Serum/Plasma Kit in combination with the QIAvac24 Plus system, a vacuum manifold that enables handling of high volumes during RNA isolation. 35 ng of total RNA were passed on to cDNA library preparation followed by small RNA high-throughput sequencing analysis on the Illumina HiSeq2000 platform. Raw sequencing reads were processed by a data analysis pipeline using different free software solutions. Seq-data was trimmed, quality checked, gradually selected for miRNAs/piRNAs and aligned to small RNA reference annotation indexes. Mapping to human reference indexes resulted in 4.8±2.8% of mature miRNAs and 1.4±0.8% of piRNAs and of 5.0±2.9% of mature miRNAs for bos taurus.
format	article
author	Melanie Spornraft Benedikt Kirchner Bettina Haase Vladimir Benes Michael W Pfaffl Irmgard Riedmaier
author_facet	Melanie Spornraft Benedikt Kirchner Bettina Haase Vladimir Benes Michael W Pfaffl Irmgard Riedmaier
author_sort	Melanie Spornraft
title	Optimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.
title_short	Optimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.
title_full	Optimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.
title_fullStr	Optimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.
title_full_unstemmed	Optimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.
title_sort	optimization of extraction of circulating rnas from plasma--enabling small rna sequencing.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/5272204a5ba445689dc74329bdfed01a
work_keys_str_mv	AT melaniespornraft optimizationofextractionofcirculatingrnasfromplasmaenablingsmallrnasequencing AT benediktkirchner optimizationofextractionofcirculatingrnasfromplasmaenablingsmallrnasequencing AT bettinahaase optimizationofextractionofcirculatingrnasfromplasmaenablingsmallrnasequencing AT vladimirbenes optimizationofextractionofcirculatingrnasfromplasmaenablingsmallrnasequencing AT michaelwpfaffl optimizationofextractionofcirculatingrnasfromplasmaenablingsmallrnasequencing AT irmgardriedmaier optimizationofextractionofcirculatingrnasfromplasmaenablingsmallrnasequencing
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Optimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.

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