White blood cells (WBCs) were isolated from approximately 3 to 4 mL whole blood samples by a density gradient centrifugation using HetaSep (STEMCell Technologies, Vancouver, Canada). Peripheral blood mononuclear cells (PBMCs) were isolated from approximately 3 to 4 mL whole blood samples by a density gradient centrifugation using Lymphoprep (STEMCell Technologies). RNA was extracted from WBC and PBMC samples using the miRNAeasy Mini kit (Qiagen, Manchester, United Kingdom). The quantity of isolated RNA was determined by spectrophotometry with an ND-1000 NanoDrop (Thermo Fisher Scientific, Waltham, MA), and quality was assessed using a Tapestation 220 (Agilent Technologies, Santa Clara, CA).
Tapestation 220
Manufactured by Agilent Technologies
Sourced in United States
The Tapestation 220 is a lab instrument used for automated analysis of DNA, RNA, and protein samples. It provides rapid size and quality assessment of samples in a user-friendly platform.
Automatically generated - may contain errors
Lab products found in correlation
Paxgene blood mirna kit, by Qiagen (6 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Qiacube, by Qiagen (6 mentions)
Nanodrop nd 1000, by Agilent Technologies (5 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (3 mentions)
Ribopure blood rna purification kit, by Thermo Fisher Scientific (2 mentions)
Hetasep, by STEMCELL (1 mentions)
Mirnaeasy mini kit, by Qiagen (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Enhanced avian hs rt pcr kit, by Merck Group (1 mentions)
Lgm 3 culture medium, by Lonza (1 mentions)
Rneasy midi kit, by Qiagen (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
8 protocols using tapestation 220
1
Isolation and Characterization of WBCs and PBMCs
2
Total RNA Extraction and cDNA Synthesis
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Total RNA from samples collected in PAXgene tubes from RT patients was extracted with the PAXgene Blood miRNA kit (Qiagen, PreAnalytiX GmbH, Hilden, Germany) using a robotic workstation Qiacube (Qiagen, Manchester, UK). The quantity of isolated RNA was determined by spectrophotometry with a ND-1000 NanoDrop and quality was assessed using a Tapestation 220 (Agilent Technologies, Santa Clara, CA, USA). cDNA was prepared from 350 ng of total RNA using the High Capacity cDNA reverse transcription kit (Applied Biosystems, FosterCity, CA, USA) according to the manufacturer’s protocol. Alternatively, total RNA was reverse-transcribed using the HS-RT100 kit (Sigma-Aldrich, L’Isle d’Abeau, France) with anchored oligo dT priming according to the manufacturer’s protocol.
3
Isolation and Quantification of Total RNA
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Total RNA was extracted with the PAXgene Blood miRNA kit (Qiagen, PreAnalytiX GmbH, Hilden, Germany) using a robotic workstation Qiacube (Qiagen, Manchester, UK). The quantity of isolated RNA was determined by spectrophotometry with a ND-1000 NanoDrop and quality was assessed using a Tapestation 220 (Agilent Technologies, Santa Clara, CA, USA).
A high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) was used to prepare cDNA from 700 ng for the in vivo irradiated samples or 200 ng for the ex vivo irradiated samples of total RNA according to the manufacturer’s protocol.
A high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) was used to prepare cDNA from 700 ng for the in vivo irradiated samples or 200 ng for the ex vivo irradiated samples of total RNA according to the manufacturer’s protocol.
4
Total RNA Extraction and cDNA Synthesis from Blood Samples
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Total RNA was extracted with the PAXgene Blood miRNA kit (Qiagen, PreAnalytiX GmbH, Hilden, Germany) from blood samples collected in PAXgene tubes from the same RT patients as enrolled in immune phenotypical analysis using a robotic workstation Qiacube (Qiagen, Manchester, UK). The quantity of isolated RNA was determined by spectrophotometry with a ND-1000 NanoDrop and quality was assessed using a Tapestation 220 (Agilent Technologies, CA, USA). cDNA was prepared from 350 ng of the total RNA using High Capacity cDNA reverse transcription kit (Applied Biosystems, FosterCity, CA, USA) according to the manufacturer’s protocol.
5
Extraction and Evaluation of RNA from Irradiated Blood
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Total RNA from blood samples exposed ex vivo to X-rays was extracted using a RiboPure™ Blood RNA Purification Kit (Thermo Fisher Scientific, Loughborough, UK). Total RNA from samples collected in PAXgene tubes from radiotherapy patients was extracted with the PAXgene Blood miRNA Kit (QIAGEN, PreAnalytiX GmbH, Hilden, Germany) using a robotic workstation Qiacube (QIAGEN, Skelton House, Lloyd St., N., Manchester M15 6SH, UK). The quantity of isolated RNA was determined by spectrophotometry with a ND-1000 NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) and quality was assessed using a Tapestation 220 (Agilent Technologies, Santa Clara, CA, USA). cDNA was prepared from 350 ng of the total RNA using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol.
6
Radiation-induced RNA extraction and analysis
7
RNA Extraction and cDNA Synthesis
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Total RNA was extracted with the PAXgene Blood miRNA kit (Qiagen, PreAnalytiX GmbH, Hilden, Germany) using a robotic workstation Qiacube (Qiagen, Manchester, UK). The quantity of isolated RNA was determined by spectrophotometry with a ND-1000 NanoDrop and quality was assessed using a Tapestation 220 (Agilent Technologies, CA, USA). cDNA was prepared from 350 ng of the total RNA using High Capacity cDNA reverse transcription kit (Applied Biosystems, FosterCity, CA, USA) according to the manufacturer’s protocol.
8
Radiation-Induced Transcriptional Changes
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Blood from nine healthy donors was collected and exposed to 0 Gy or 2 Gy (10 mL of blood each, dose rate 0.5 Gy/min). After irradiation, the peripheral blood mononuclear cells (PBMCs) were isolated using Histopaque-1077 (Sigma Aldrich, Poole, Dorset, UK) and maintained in LGM-3 culture medium (Lonza, Slough, UK) at 2 × 106 cells ml/mL for 24 h at 37 °C in a humidified 5% CO2 atmosphere. After 24 h, the PBMCs were pooled in groups of three donors each, and the RNA was extracted using the RNeasy Midi kit (Qiagen, Manchester, UK). The quantity of isolated RNA was determined by spectrophotometry with a ND-1000 NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) and quality was assessed using a Tapestation 220 (Agilent Technologies, CA, USA). Venous blood was taken at the Centre for Radiation, Chemical and Environmental Hazards Public Health England (Chilton, UK) with informed consent and the ethical approval of the West Midlands Solihull Research Ethics Committee (REC 14/WM/1182).
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