RNA was extracted from the whole blood samples using PAXgene Blood miRNA Kit (Qiagen, Hombrechtikon, Switzerland) according to a previously established protocol (Unger et al. 2016 (link)). RNA-concentrations and RNA Integrity Numbers (RIN) were measured with a Fragment Analyzer and the Standard Sensitivity RNA Analysis Kit (Advanced Analytical, Heidelberg, Germany). The extracted RNA was stored at -80 °C.
Standard sensitivity rna analysis kit
Manufactured by Agilent Technologies
Sourced in Germany, United States
The Standard Sensitivity RNA Analysis Kit is a laboratory tool designed for the analysis of RNA samples. It provides the necessary reagents and protocols to enable the detection and quantification of RNA molecules within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Fragment analyzer, by Agilent Technologies (10 mentions)
Qubit rna br assay kit, by Thermo Fisher Scientific (4 mentions)
Paxgene blood mirna kit, by Qiagen (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Hiseq 3000, by Illumina (2 mentions)
Dna free dna removal kit, by Thermo Fisher Scientific (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Spectrum plant total rna kit, by Merck Group (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rapidout dna removal kit, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Qubit br assay, by Thermo Fisher Scientific (1 mentions)
Truseq stranded mrna kit, by Illumina (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (1 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (1 mentions)
Miscript primer assay, by Qiagen (1 mentions)
Quantifluor dsdna system, by Promega (1 mentions)
Truseq stranded total rna library preparation, by Illumina (1 mentions)
17 protocols using standard sensitivity rna analysis kit
1
Blood RNA Extraction Using PAXgene Kit
2
Transcriptome Analysis of Larval Samples
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For the RNA sequencing, total RNA was extracted from the pools of five larvae using the Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The genomic DNA was removed from the samples using the RapidOut DNA removal Kit (Thermo Scientific, Thermo Fisher Scientific) and the purity and the concentration of the samples were measured with Qubit RNA BR Assay Kit (Invitrogen, Thermo Fisher Scientific). The RNA integrity was analyzed with the Fragment Analyzer (Advanced Analytical Technologies, Iowa, USA) using the Standard Sensitivity RNA Analysis Kit (Advanced Analytical Technologies) and the PROSize® 2.0 Data Analysis Software (Advanced Analytical Technologies). The RNA samples with an integrity number (RIN) >9 were used in the transcriptome analysis. Finally, two RNA samples were combined to achieve sufficient concentration for the transcriptome analysis.
For the qPCR analysis of crp2-1, crp2-2 and crp3 expression in WT and mutant crp2tpu6 larvae and of crp3 expression in crp3 SB morpholino and RC morpholino injected larvae, total RNA was extracted from the pools of 3–5 larvae using the Qiagen RNeasy Plus Mini Kit with genomic DNA elimination step (Qiagen) according to the manufacturer’s instructions. The purity and the concentration of the samples were measured with NanoDrop 2000 Spectrophotometer (Thermo Scientific, Thermo Fisher Scientific).
For the qPCR analysis of crp2-1, crp2-2 and crp3 expression in WT and mutant crp2tpu6 larvae and of crp3 expression in crp3 SB morpholino and RC morpholino injected larvae, total RNA was extracted from the pools of 3–5 larvae using the Qiagen RNeasy Plus Mini Kit with genomic DNA elimination step (Qiagen) according to the manufacturer’s instructions. The purity and the concentration of the samples were measured with NanoDrop 2000 Spectrophotometer (Thermo Scientific, Thermo Fisher Scientific).
3
RNA Extraction and Illumina Sequencing
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RNA was extracted from snap-frozen tissue with the RNeasy Micro Kit (Qiagen) quantified by the Qubit BR assay (Thermo Fisher Scientific) and quality was assessed by Fragment Analyzer (Advanced Analytical) using the Standard Sensitivity RNA Analysis Kit (Advanced Analytical). Libraries were prepared without size exclusion with the TruSeq Stranded mRNA kit (Illumina). Qubit double-stranded DNA high sensitivity (Thermo Fisher Scientific) and Fragment Analyzer NGS (Advanced Analytical) kits were used for library quality control. Finally, the samples were sequenced on a HiSeq3000 (Illumina), 1x100 bp on 4.5 lanes.
4
RNA-Seq Analysis of E. coli Transcriptome
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RNA sequencing (RNA-Seq) was performed and analyzed as recently described (18 (link)). Briefly, overnight cultures were diluted to OD600 = 0.1, grown for 1 h, and treated or not with 10 μM TAT-RasGAP317-326 for 1 h. RNA was extracted with RNeasy Plus minikit (Qiagen), and any remaining DNA was removed using DNA-free DNA Removal Kit (Invitrogen, Carlsbad, CA). Integrity of the resulting RNA samples was verified by Standard Sensitivity RNA Analysis kit (Advanced Analytical, Ankeny, IA) with the Fragment Analyser Automated CE System (Labgene Scientific, Châtel-Saint-Denis, Switzerland). Preparation of the libraries and Illumina HiSeq platform (1 × 50 bp) sequencing were performed by Fasteris (Plan-les-Ouates, Switzerland). Reads were then trimmed and mapped to genome of E. coli K-12 MG1655. Normalized expression values were calculated as reads per kilobase of transcript per million mapped reads (RPKM) with edgeR (50 (link)).
5
Chemically Modified mRNA Synthesis
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Chemically modified mRNA (mRNA) was synthesized as previously described by Kormann et al. [42 (link)]. In brief, the respective pDNA templates were subjected to in vitro transcription using T7 RNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) with a predefined mix of natural and chemically modified ribonucleotides. To enhance RNA stability, a m7G cap structure was added to the 5′ end of the transcript, while the 3′ end was subjected to enzymatic polyadenylation of ~120 nucleotides. The mRNA product was purified by ammonium-acetate precipitation and was re-suspended in water at the desired concentration. Standard 260/280 nm ratio was determined on a NanoDrop2000C spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and mRNA integrity and size were determined using a Standard Sensitivity RNA Analysis Kit on a Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA, USA).
6
Equine Whole Blood RNA Extraction
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RNA was extracted according to an optimized protocol for long-term stored equine whole blood samples [35 (link)]. Prior to RNA-extraction, a synthetic spike-in control—cel-miR-39-3p [5.6×108 copies, Cel_miR-39_1 miScript Primer Assay, Qiagen, Hombrechtikon, Switzerland] was added to each sample. This exogenous control allowed for the monitoring of RNA-extraction efficiency.
RNA concentration was measured using a Qubit 2.0 fluorimeter, in combination with the Qubit™ RNA BR assay kit [ThermoFisher, Reinach, Switzerland]. The RNA Integrity Number (RIN) was assessed using a Fragment Analyzer and the Standard Sensitivity RNA Analysis Kit [Advanced Analytical, Heidelberg, Germany]. The extracted RNA was stored at—80°C for further analysis.
RNA concentration was measured using a Qubit 2.0 fluorimeter, in combination with the Qubit™ RNA BR assay kit [ThermoFisher, Reinach, Switzerland]. The RNA Integrity Number (RIN) was assessed using a Fragment Analyzer and the Standard Sensitivity RNA Analysis Kit [Advanced Analytical, Heidelberg, Germany]. The extracted RNA was stored at—80°C for further analysis.
7
Comprehensive RNA-seq Library Preparation and Sequencing
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NGS-Sequencing and library preparation was conducted at the NGS- Integrative Genomics Core Unit (NIG), Institute of Human Genetics, University Medical Center Göttingen (UMG).
Quality and integrity of RNA was assessed with the fragment analyzer from Advanced Analytical by using the standard sensitivity RNA analysis Kit (DNF-471). All samples selected for sequencing exhibited an RNA integrity number over 8. RNA-seq libraries were performed using 200 ng total RNA of a non-stranded RNA Seq, massively-parallel mRNA sequencing approach from Illumina (TruSeq stranded total RNA Library Preparation, Illumina). Libraries were prepared on the automation (Beckman Coulter’s Biomek FXP workstation). For accurate quantitation of cDNA libraries a fluorometric based system, the QuantiFluor™dsDNA System from Promega were used. The size of final cDNA libraries was determined by using the dsDNA 905 Reagent Kit (fragment analyzer from Advanced Bioanalytical) exhibiting a sizing of 300 bp in average. Libraries were pooled and sequenced on the Illumina HiSeq 4000 (SE; 1 × 50 bp; 30–35 Mio reads/sample). Sequence images were transformed with Illumina software BaseCaller to BCL files, which was demultiplexed to fastq files with bcl2fastq v2.20.0.422. The quality check was done using FastQC.
Quality and integrity of RNA was assessed with the fragment analyzer from Advanced Analytical by using the standard sensitivity RNA analysis Kit (DNF-471). All samples selected for sequencing exhibited an RNA integrity number over 8. RNA-seq libraries were performed using 200 ng total RNA of a non-stranded RNA Seq, massively-parallel mRNA sequencing approach from Illumina (TruSeq stranded total RNA Library Preparation, Illumina). Libraries were prepared on the automation (Beckman Coulter’s Biomek FXP workstation). For accurate quantitation of cDNA libraries a fluorometric based system, the QuantiFluor™dsDNA System from Promega were used. The size of final cDNA libraries was determined by using the dsDNA 905 Reagent Kit (fragment analyzer from Advanced Bioanalytical) exhibiting a sizing of 300 bp in average. Libraries were pooled and sequenced on the Illumina HiSeq 4000 (SE; 1 × 50 bp; 30–35 Mio reads/sample). Sequence images were transformed with Illumina software BaseCaller to BCL files, which was demultiplexed to fastq files with bcl2fastq v2.20.0.422. The quality check was done using FastQC.
8
Long-term Equine Blood RNA Sequencing
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Whole blood samples used in this study were stored for long term (9‐11 years) for horses in the ES regression and progression groups and for short term (6 weeks) for control horses at −80°C before the analysis for this study. Samples were handled according to a previously established protocol for long‐term stored equine whole blood.25 The blood was gently thawed on ice and transferred to a PAXgene blood RNA tube 16 hours before the start of RNA extraction with a PAXgene Blood miRNA Kit (Qiagen, Hombrechtikon, Switzerland). The RNA concentrations were measured with Qubit RNA BR assay kit (ThermoFisher, Basel, Switzerland). The RNA integrity number (RIN) was assessed using Fragment Analyzer with the Standard Sensitivity RNA Analysis Kit (Advanced Analytical, Heidelberg, Germany). A total of 14.6‐54.6 ng of small RNA was used for stranded single‐end library preparation (NEBNext Multiplex Small RNA Library Prep Set for Illumina) and sequenced (HiSeq3000, Illumina).
9
Comprehensive RNA-Seq Analysis of Isolated Hepatic Tissue
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Quality of total isolated hepatic RNA was tested with Standard Sensitivity RNA Analysis Kit (Advanced Analytical Technologies) and library prepared with TruSeq® Stranded mRNA HT (Illumina). Libraries were validated with Standard Sensitivity NGS Fragment Analysis Kit 1‐ 6,000bp (Advanced Analytical Technologies), pooled in equimolar concentrations, diluted and denatured according to Illumina guidelines. Samples were sequenced with NextSeq 500/550 using High Output v2 kit (Illumina) to average 9 million reads. Base calling were done by bcl2fastq from Illumina. Reads were mapped to mouse genome version mm10 with HiSat2 v.2.0.1‐beta and counts generated using featurecounts (version 1.4.4) and TPMs were generated using Sailfish v. 0.7.6. Normalized counts were analyzed with Array Studio software (OmicSoft, Cary, NC, USA) and raw p‐values and False Discovery Rates (FDR) probability calculated. The set of genes with fold changes ≥ ±2 and p‐raw ≤0.05 were used for MetaCore pathway enrichment analysis.
10
Robust Total RNA Extraction from Fruit
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Total RNA of 24 samples was extracted from 100 mg of fruit flesh using a mortar and pestle along with the SpectrumTM plant total RNA kit, following the manufacturer’s instructions (Sigma Aldrich, Saint Louis, MO, USA). The quantification was performed using a Qubit® 2.0 fluorometer and a QubitTM RNA BR assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The quality control process of each RNA sample extracted was made using a Fragment Analyzer™ automated CE system (Analytical Advanced Technologies, Ames, IA, USA), 0.1–0.8 μg of total RNA were analyzed using a Standard Sensitivity RNA analysis kit (Advanced Analytical Technologies) following the manufacturer’s recommendations, and finally, ProSize 2.0 software (Analytical Advanced Technologies) was used to determine the RNA quality, considering an RQN value of 8.0 as useful for library construction and sequencing.
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