Blood samples (2.5 ml) were drawn using PaxGene Blood RNA tubes (PreAnalytix GmbH, Hombrechtikon, Switzerland) and total RNA was then isolated as described in a previous publication [11 (link)]. The integrity of the purified RNA was accessed by 2100 Bioanalyzer RNA 6000 Nano Chips (Agilent Technologies, Inc., Santa Clara, CA, USA) and the quantity of RNA was assessed by NanoDrop 1000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Inc. Waltham, MA, USA). All RNA samples were assessed by RNA integrity number ≥7·0 and 28S:18S rRNA≥1.0.
2100 bioanalyzer rna 6000 nano chips
Manufactured by Agilent Technologies
Sourced in United States
The 2100 Bioanalyzer RNA 6000 Nano Chips are a lab equipment product designed to analyze the quality and quantity of RNA samples. The chips provide a platform for the automated electrophoretic separation and detection of RNA molecules in a microfluidic system.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Paxgene blood rna tube, by Preanalytix (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Scriptseqtm v2 rna seq library preparation kit, by Illumina (1 mentions)
Hiseq system, by Illumina (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Ribo zero rrna removal kit, by Illumina (1 mentions)
Scriptseq v2 rna seq library preparation kit, by Illumina (1 mentions)
Miseq system, by Illumina (1 mentions)
Purelink mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Rneasy protect animal blood kit, by Qiagen (1 mentions)
Rna clean concentrator kit, by Zymo Research (1 mentions)
Paxgene blood rna kit, by Preanalytix (1 mentions)
8 protocols using 2100 bioanalyzer rna 6000 nano chips
1
RNA Isolation from Blood Samples
2
Transcriptomic Profiling of Pneumococcus
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Total RNA was isolated at different time points from S. pneumoniae R6, CCRI-8970 and CCRI-21487 grown in BHI using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. The RNAs were treated with DNase I (Ambion) to avoid any DNA contamination. RNAs were quantified using 2100 BioAnalyzer RNA6000 Nano chips (Agilent) and 1 µg of total RNA was treated with Ribo-ZeroTM Magnetic Kit for Gram-Positive Bacteria (Epicentre). The rRNA-depleted samples were purified using RNeasy MinElute Cleanup kit (Qiagen). RNA-seq libraries were produced from 50 ng of rRNA-depleted samples using the ScriptSeqTM v2 RNA-Seq Library Preparation Kit (Epicentre). The libraries were analysed using 2100 BioAnalyser High Sensitivity DNA Chips (Agilent) and quantified by QuantiFluor. The libraries were pooled, diluted to 8 pM and sequenced on an Illumina HiSeq system using a 101 bp paired-ends reads protocol.
3
RNA Isolation and Quality Assessment
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Peripheral whole blood (2.5 ml) was collected in PaxGene Blood RNA tubes (PreAnalytix GmbH, Hombrechtikon, Switzerland). Total RNA was then isolated as described previously (11 ). RNA quality was accessed using 2100 Bioanalyzer RNA 6000 Nano Chips (Agilent Technologies, Inc., Santa Clara, CA, USA). All the samples for microarray analysis met the following quality criteria: RNA integrity number ≥7.0 and 28S:18S rRNA ≥1.0. RNA quantity was determined by a NanoDrop 1000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
4
RNA-Seq Analysis of S. pneumoniae Mutant
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Total RNA was isolated from S. pneumoniae R6Δspr0058 and R6 WT grown to mid-log phase in BHI using the Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNAs were quantified using 2100 BioAnalyzer RNA6000 Nano chips (Agilent) and 1 μg of total RNA was treated with Ribo-Zero™ rRNA Removal Kits (Epicentre). RNA-seq libraries were produced from 50 ng of rRNA-depleted samples using the ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre). The libraries were analyzed using 2100 BioAnalyser High Sensitivity DNA chips and quantified by PicoGreen. The libraries were pooled, diluted to 8 pM and sequenced on an Illumina MiSeq system using a 250 bp paired-ends reads protocol. Sequence reads from each strain were filtered based on quality score using Trimmomatic [73 (link)] and aligned to the genome of S. pneumoniae R6 using the software bwa with default parameters [74 (link)]. A total of 3,843,210 and 3,245,555 reads derived from S. pneumoniae R6 WT and R6Δspr0058 mapped to the S. pneumoniae R6 reference genome, respectively. The maximum number of mismatches was 4 and the seed length was 32. Transcripts were assembled from the alignment files by using the Cufflinks pipeline [75 (link)]. Differential gene expression was computed with CuffDiff and genes with a false-discovery rate-adjusted p-value ≤ 0.05 were considered as differentially expressed.
5
RNA Extraction from Nematodes and Mouse Blood
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For the nematodes, 1 ml TRIzol (Thermo Fisher Scientific) was added to each worm pellet and briefly grinded with a pestle. The mixture was then frozen in liquid nitrogen and thawed at room temperature three times. RNA of adult worms was extracted with the standard TRIzol protocol followed by a separation of large and small RNAs with the PureLink miRNA Isolation Kit (Thermo Fisher Scientific). RNA of dauer/infective larvae was extracted the same as adult RNA but the small RNA fraction contained a lot of large RNAs and was reseparated into large and small RNA fractions using the RNA Clean & Concentrator kit (Zymo Research). For the mouse blood, the total RNA (including short RNAs) from whole blood of infected and uninfected rats was extracted with the RNeasy Protect Animal Blood kit (Qiagen) according to the manufacturer’s protocol. Subsequently, the large RNA and small RNA fractions were obtained with the RNA Clean & Concentrator kit (Zymo Research). The quality and quantity of the extracted RNA fractions was assessed using 1% Agarose gels, 2100 Bioanalyzer RNA 6000 Nano chips (Agilent Technologies), and Qubit RNA BR Assays (ThermoFisher Scientific) according to the manufacturer’s protocol. The RNA was stored at –80 °C until library preparation.
6
Peripheral Blood RNA Isolation
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Peripheral whole blood (2.5 ml) was collected in PaxGene Blood RNA tubes (PreAnalytiX GmbH; Qiagen). Total RNA was then isolated using the PaxGene Blood RNA kit (PreAnalytiX GmbH; Qiagen) following the manufacturer's protocol. RNA quality was assessed using a 2100 Bioanalyzer RNA 6000 Nano Chips (Agilent Technologies, Inc.), according to the manufacturer's protocol. All samples for microarray analysis met the following quality criteria: RNA Integrity number ≥7.0 and 28S:18S ribosomal RNA ≥1.0. RNA quantity was determined using a NanoDrop 1000 UV–Vis spectrophotometer (Thermo Fisher Scientific, Inc.).
7
Transcriptional Analysis of Cryptosporidium-Infected Organoids
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Organoids derived from two independent donors were microinjected with oocyst or control media. Total RNA was extracted from collected organoids at 24 and 72 hr post-injection using RNeasy Micro Kit (Qiagen) according to manufacturer’s protocol including DNaseI treatment. Quality and quantity of isolated RNA was checked and measured with Bioanalyzer 2100 RNA Nano 6000 chips (Agilent). Libraries were prepared as described previously47 (link) and sequenced on an Illumina NextSeq500 by using 75-bp paired-end sequencing. Paired-end reads from Illumina sequencing were aligned to the human transcriptome genome and C. parvum transcriptome genome (Iowa strain) by BWA48 (link). DeSeq (v1.18.0) was used for read normalization and differential expression analysis. Gene set enrichment analysis (GSEA) was performed using gene lists for type I interferon response and regulation against normalized RNA-seq reads of injected SI and lung organoids using GSEA software v3.0 beta2.
8
Transcriptional Analysis of Cryptosporidium-Infected Organoids
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