RNA samples were extracted using the IllustraRNAspin Mini kit (GE Healthcare). Following extraction, RIN (RNA Integrity Number) was >9 (BioAnalyser, Agilent RNA Nano Kit). RNA samples (500 ng) were reverse transcribed with the IlluminaTotalPrep RNA Amplification Kit (Ambion) and copy RNA (cRNA) was generated with 14 hr in vitro transcription reactions and checked at the BioAnalyser. Washing, staining, and hybridization were performed according to standard Illumina protocols. cRNA samples were then hybridized to IlluminaBeadChip Array MouseRefSeq-8 v2. BeadChips were scanned with BeadArray™ Reader in channel 2. The data have been uploaded on the Dryad Digital Repository (Zambrano et al., 2016 ). Experiments were repeated twice.
Agilent rna nano kit
Manufactured by Agilent Technologies
Sourced in United States
The Agilent RNA Nano Kit is a laboratory equipment used for the analysis and quantification of RNA samples. The kit provides the necessary reagents and consumables to perform size-based separation and detection of RNA on the Agilent Bioanalyzer platform.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Ribo zero rrna removal kit, by Illumina (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Illustra rnaspin mini kit, by GE Healthcare (1 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Agilent rna pico kit, by Agilent Technologies (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Direct zol rna microprep kit, by Zymo Research (1 mentions)
Recoverall total nucleic acid isolation kit for ffpe, by Thermo Fisher Scientific (1 mentions)
Humanht 12 v4 beadchip, by Illumina (1 mentions)
Globinclear human kit, by Thermo Fisher Scientific (1 mentions)
Paxgene blood mirna kit, by Preanalytix (1 mentions)
Rneasy minielute kit, by Qiagen (1 mentions)
Biophotometer, by Eppendorf (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna ffpe kit, by Qiagen (1 mentions)
11 protocols using agilent rna nano kit
1
Mouse Transcriptome Profiling by Illumina
2
Quantitative Analysis of miRNA-155 in Pediatric Glioma
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Formalin-Fixed Paraffin-Embedded (FFPE) Tumors of Pediatric high-grade glioma were cut using a microtome into 10 μm slices. The Recover All Total Nucleic Acid Isolation Kit for FFPE Tissues (Fisher Scientific, Hampton, NH USA) was used for the total RNA enriched with a miRNA fraction isolation according to the manufacturer’s protocol. The quality of the obtained RNA was determined using micro-capillary electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA USA) and an Agilent RNA Nano kit (Agilent Technologies, Santa Clara, CA USA). The purity and concentration of RN A obtained from the FFPE was assessed using spectrophotometry (NanoDrop 2000c, ThermoFisher Scientific, Waltham, MA USA). Concentration of hsa-miR-155-5p was determined using enzyme immunoassay for miRNA quantification hsa-miR-155-5p miREIA (BioVendor, Brno, Czech Republic) according to the manufacturer’s protocol.
3
Assessing Tumor RNA Quality and Integrity
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The concentration of the tumor RNA samples was determined using the Qubit RNA HS Assay Kit on a Qubit 4 fluorometer (both Thermo Fisher Scientific). The RNA was assessed for quality and integrity using the profiles generated by an Agilent Bioanalyzer. For the tumor tissue, the RNA was diluted to meet the recommended range and assessed using an Agilent RNA Nano Kit; the plasma RNA was directly assessed using an Agilent RNA Pico Kit according to the manufacturer’s instructions (both Agilent Technologies, Santa Clara, CA, USA).
The tumor RNA was also assessed for gDNA contamination by a GAPDH RT-qPCR (cutoff Ct 30) using a 100 ng tumor RNA template, 2× Power SYBR™ Green PCR Master Mix (Applied Biosystems, Foster, KY, USA) and 4µM of forward and reverse primers (Invitrogen, 5’-GAAGGTGAAGGTCGGAGTC-3’ and 5’-GAAGATGGTGATGGGATTTC-3’) in a total reaction mix of 25 µL. The thermocycling conditions were as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min with data collection. The cycling was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems). The Ct values were analyzed using the StepOne Software (v2.3) with auto settings.
The tumor RNA was also assessed for gDNA contamination by a GAPDH RT-qPCR (cutoff Ct 30) using a 100 ng tumor RNA template, 2× Power SYBR™ Green PCR Master Mix (Applied Biosystems, Foster, KY, USA) and 4µM of forward and reverse primers (Invitrogen, 5’-GAAGGTGAAGGTCGGAGTC-3’ and 5’-GAAGATGGTGATGGGATTTC-3’) in a total reaction mix of 25 µL. The thermocycling conditions were as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min with data collection. The cycling was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems). The Ct values were analyzed using the StepOne Software (v2.3) with auto settings.
4
RNA Extraction from FFPE and Frozen Tissues
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RNA from FFPE samples of RU02135, of the controls C13-1336 and C12-841, and of RU02092, who is affected by a homozygous PNLDC1 variant (GenBank: NM_173516.2; c.607−2A>T), was extracted using Recover All Total Nucleic Acid Isolation Kit for FFPE (Invitrogen, Cat. #AM1975). RNA from snap-frozen testicular tissues of the controls SSC764, M2350, and M2676 and individuals M1400, M2546, and M2548 was extracted using Direct-zol RNA Microprep kit (Zymo Research, Cat. #R2062).
The quantity and quality of the isolated RNA were assessed with Qubit RNA High Sensitivity kit (Inivitrogen, Cat. #Q32852) and Agilent RNA Nano kit (Agilent, Cat. #55067-1512), respectively.
The quantity and quality of the isolated RNA were assessed with Qubit RNA High Sensitivity kit (Inivitrogen, Cat. #Q32852) and Agilent RNA Nano kit (Agilent, Cat. #55067-1512), respectively.
5
RNA-seq Library Preparation and Sequencing
6
RNA-seq Library Preparation and Sequencing
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RNA integrity was tested with Bioanalyzer (Agilent RNA Nano Kit) and ribosomal RNA was removed using Ribo-Zero rRNA Removal Kit (Illumina) prior to library preparation. Strand-specific libraries were prepared with NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®. After amplification and size selection with Agencourt AMPure XP beads (Beckmann Coulter) their size-distributions were determined with Bioanalyzer. Equimolar pools of libraries were sequenced with Illumina HiSeq2000 (50bp, single end). We retrieved on average 25 mio reads per sample, of which 19 mio reads were uniquely mapped to the reference genome (NCBI37/mm9).
7
Globin mRNA Removal from Whole Blood RNA
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RNA was extracted from peripheral whole blood samples using the PAXgene Blood miRNA kit (PreAnalytix GmbH, Switzerland). The extractions were performed according to the manufacturer’s protocol. The RNeasy MiniElute kit (QIAGEN, Manchester) was used to obtain the required concentration and volume for the Globin mRNA reduction procedure.
Globin mRNA was removed from the RNA samples using the Human GLOBINclear kit (Ambion Inc., Texas, USA). The purity and the concentration of the globin-cleared samples were assessed using the Nano-drop ND-1000 spectrophotometer (Willmington, USA). The samples were stored at −20°C, according to the GLOBINclear manufacturer’s protocol [68 ]. The quality of all samples was analysed with the Agilent 2100 Bioanalyzer using the Agilent RNA Nano kit (Agilent, Santa Clara, USA). Samples with a RNA integrity number (RIN) of above seven were used for whole genome microarray using the Illumina HumanHT-12 v4 BeadChip. Both techniques were performed at Cambridge Genomic Services (Cambridge, UK).
Globin mRNA was removed from the RNA samples using the Human GLOBINclear kit (Ambion Inc., Texas, USA). The purity and the concentration of the globin-cleared samples were assessed using the Nano-drop ND-1000 spectrophotometer (Willmington, USA). The samples were stored at −20°C, according to the GLOBINclear manufacturer’s protocol [68 ]. The quality of all samples was analysed with the Agilent 2100 Bioanalyzer using the Agilent RNA Nano kit (Agilent, Santa Clara, USA). Samples with a RNA integrity number (RIN) of above seven were used for whole genome microarray using the Illumina HumanHT-12 v4 BeadChip. Both techniques were performed at Cambridge Genomic Services (Cambridge, UK).
8
Comprehensive RNA Quality Assessment
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RNA integrity was checked using Bioanalyzer 2100 (Agilent Technologies Inc.) and Agilent RNA Nano kit. RIN values of RNA ranged between 6 and 8.6. Only samples with RIN≥ 6 were used in downstream applications. Concentration and purity of RNA were measured using spectrophotometry (Biophotometer with Hellma TrayCell, Eppendorf). 260/280 ratio of all RNA samples ranged between 1.8–2.2. All samples were stored at −80°C.
9
Transcriptomics of Lung Adenocarcinoma Cell Lines
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Total RNA purified from seven lung adenocarcinoma cell lines (PC-7, PC-9, H1975, H2228, VMRC-LCD, LC2/ad, and A549) were used. We purchased 14 total RNAs from seven organs, liver, kidney, skeletal muscle, pancreas, colon, heart, and lung, derived from two individuals (BioChain Institute). We also purchased two genomic DNAs derived from them. We conducted a quality check of the total RNA using the Agilent RNA Nano Kit.
10
RNA Quality Assessment and Storage
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RNA integrity was checked using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and Agilent RNA Nano kit. RIN values of RNA ranged between 6 and 8.6. Only samples with RIN ≥ 6 were used in downstream applications. Concentration and purity of RNA were measured using spectrophotometry and Biophotometer with Hellma TrayCell (Eppendorf, Hamburg, Germany). 260/280 ratio of all RNA samples ranged between 1.8 and 2.2. All samples were stored at −80 °C.
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