Blood was collected into Tempus Blood RNA tubes (Applied Biosystems) and the RNA was extracted using the MagMAX for Stabilized Blood Tubes RNA Isolation Kit, compatible with Tempus Blood RNA tubes (ThermoFisher Scientific). RNA quality was assessed using a TapeStation 4200 (Agilent) and then 200 nanograms of total RNA was used as input for cDNA synthesis and library preparation using the KAPA RNA HyperPrep kit with RiboErase (HMR) Globin (Roche) according to the manufacturer’s instructions. Libraries were validated by capillary electrophoresis on a TapeStation 4200 (Agilent), pooled at equimolar concentrations, and sequenced with PE100 reads on an Illumina NovaSeq 6000, yielding ~60 million reads per sample on average.
Tempus blood rna tube
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
The Tempus Blood RNA Tubes are designed to stabilize and preserve RNA in whole blood samples for subsequent analysis. The tubes contain a proprietary reagent that immediately lyses blood cells and stabilizes the RNA, allowing for reliable RNA extraction and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Tempus spin rna isolation kit, by Thermo Fisher Scientific (52 mentions)
Magmax for stabilized blood tubes rna isolation kit, by Thermo Fisher Scientific (10 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (8 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (7 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (7 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (7 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (6 mentions)
Ht 12 v4 expression beadchip, by Illumina (6 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (5 mentions)
Humanht 12 v4 expression beadchip, by Illumina (5 mentions)
Rnalater, by Thermo Fisher Scientific (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Abi prism 6100 nucleic acid prepstation, by Thermo Fisher Scientific (5 mentions)
Bioanalyzer, by Agilent Technologies (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Vacuette, by Greiner (4 mentions)
Rna stabilization solution, by Thermo Fisher Scientific (4 mentions)
Tempus 12 port rna isolation kit, by Thermo Fisher Scientific (4 mentions)
191 protocols using tempus blood rna tube
1
Bulk RNA-seq from Tempus Blood Tubes
2
Blood Sample Preparation and RNA Extraction
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Example 2
This example illustrates preparation of biological samples, including RNA from blood samples.
In these investigations, whole blood and nasopharyngeal samples were collected for virus-specific PCR and high-throughput sequencing. Additionally, a blood sample was collected in a Tempus™ Blood RNA Tube (Applied Biosystems, Carlsbad, Calif.) and stored at −80° C. for subsequent gene expression analysis.
Total RNA was isolated from whole blood collected in Tempus™ Blood RNA Tubes (Applied Biosystems, Carlsbad, Calif.) according to the manufacturer's instructions. RNA quality was determined by gel-chip image (showing 28S, 18S and SS bands) and RNA integrity number (RIN, generally a >7 RIN indicates good quality RNA) using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, Calif.). All but 3 of the RNA preparations had RIN scores ≥7.0. Total RNA concentration was obtained from an absorbance ratio at 260 nm and 280 nm using a NanoDrop ND-100 spectrometry instrument (NanoDrop Inc., Wilmington, Del.).
3
Blood RNA Extraction and WBC Profiling
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Blood samples were collected in the quarantine nurseryat ~ 27 days of age, using Tempus Blood RNA Tubes (Thermo Fisher Scientific, USA) and then stored at − 80 °C until RNA extraction. The RNAs were isolated using Preserved Blood RNA Purification Kit I (Norgen, Canada) according to the manufacturer’s instructions. The RNA integrity number (RIN) of each extracted RNA was assessed by the 2100 Bioanalyzer (Agilent Technologies, USA) using the Eukaryote total RNA 6000 Nano kit. The RIN score was on average 7.9 and ranged from 4.1 to 9.9 (Table 2 ). WBC differentials were quantified on whole blood samples in K2 ethylenediaminetetraacetic acid (EDTA) tubes (Thermo Fisher Scientific, USA) taken at the same time, using the flow cytometry-based hematology analyzer (ADVIA®2120i Hematology System, Simens Healthineers, Germany) according to the manufacturer’s instructions [59 (link)]. The log2 transformed proportion of each WBC type was used to adjust gene expressions levels for blood cell composition (see later).
4
Whole Blood RNA Collection Protocol
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Fasting whole blood samples were collected from each patient during clinical visits. Peripheral blood (3 mL per patient) was collected in Tempus™ Blood RNA Tubes containing 6 mL of Stabilizing Reagent (Thermo Fischer Scientific) and subsequently frozen at − 80 °C for long-term storage.
5
DNA Isolation from Blood Samples
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Sample collection was carried out using Tempus™ Blood RNA Tubes obtained from ThermoFisher. Samples were stored at room temperature in the hospital for a maximum of 5 days and subsequently transported to the Molecular Genetic Laboratory (Department of Molecular Biology, Semmelweis University) and were kept at −20 °C until further processing.
DNA isolation was initiated by adding 450 µL cell proteinase K buffer (0.1 M NaCl, 0.01 M Tris-HCl pH = 8, 0.5% SDS, 0.2 mg/mL proteinase K) to 800 µL of the sample, followed by incubation at 56 °C overnight. Proteins were then precipitated using saturated NaCl and removed by centrifugation. DNA was isolated from the supernatant using the standard ethanol/isopropanol precipitation method. Precipitated DNA was redissolved in 0.5× TE (0.005 M Tris-HCl, pH = 8 and 0.5 mM EDTA). DNA concentrations were measured with a Nanodrop1000 spectrophotometer. The average DNA concentration of the samples was 140 ng/µL (20–572 ng/µL).
DNA isolation was initiated by adding 450 µL cell proteinase K buffer (0.1 M NaCl, 0.01 M Tris-HCl pH = 8, 0.5% SDS, 0.2 mg/mL proteinase K) to 800 µL of the sample, followed by incubation at 56 °C overnight. Proteins were then precipitated using saturated NaCl and removed by centrifugation. DNA was isolated from the supernatant using the standard ethanol/isopropanol precipitation method. Precipitated DNA was redissolved in 0.5× TE (0.005 M Tris-HCl, pH = 8 and 0.5 mM EDTA). DNA concentrations were measured with a Nanodrop1000 spectrophotometer. The average DNA concentration of the samples was 140 ng/µL (20–572 ng/µL).
6
RNA Isolation and Sequencing Protocol
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RNA was isolated using the Tempus Spin RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA) from peripheral blood taken in Tempus Blood RNA tubes (Thermo Fisher Scientific) according to the manufacturer’s recommendations. First-strand reverse transcription was carried out by SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). Reverse transcription PCRs (RT-PCRs) amplifying the variant-containing exons along with at least two adjacent exons were designed individually (list of cDNA primers is given in S2 Table ). Amplification products were visualized on 1% agarose gel next to Hyper Ladder 1 kb DNA sizing standard (Bioline, London, UK) and subsequently sequenced by conventional Sanger sequencing method on ABI3130 Genetic Analyzer using the BigDye v.1.1 Kit (Thermo Fisher Scientific). Sequencing was done for the whole RT-PCR product without separation of the respective bands to compare peak intensities of normal and aberrantly spliced products. Where it was necessary to remove the interfering predominant normal alternative splice product, fragments of different sizes were cut out and cleaned from the gel by Monarch Gel Extraction Kit (New England Biolabs, Ipswich, MA) and the purified product was sequenced as above.
7
Quantitative TCR Repertoire Sequencing
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The α and β chains of the TCR repertoire of 29 participants were sequenced using a method that starts with total RNA isolated from unfractionated whole blood, collected in Tempus Blood RNA tubes (Thermofisher #4342792) using the manufacturer’s protocol for RNA extraction. The pipeline introduces unique molecular identifiers attached to individual cDNA molecules to provide a quantitative and reproducible method of library preparation. Full details for both the experimental TCRseq library preparation and the subsequent computational analysis (V, J, and CDR3 annotation) using Decombinator are published in Oakes et al., 2017b (link); Uddin et al., 2019a (link).
8
Dynamics of PRRSV Infection in Pregnant Gilts
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A detailed description of the animal experiment is available in Ladinig et al [5 (link)]. Briefly, 114 pregnant (gestation day 85±1) Landrace gilts, confirmed seronegative for PRRSV, were inoculated with 1x105 TCID50 type 2 PRRSV isolate NVSL 97–7895 on experimental day 0 (D0). Blood samples for transcriptomic analyses were collected into Tempus blood RNA tubes (Thermo Fisher Scientific, Waltham, MA, USA) on D0, D2, and D6 post-inoculation. Heparinized blood samples for flow cytometry [10 (link)] and serum samples for PRRSV quantification and cytokine analysis [11 (link)] were also collected. Gilts were humanely euthanized and necropsied at D21 (gestation day 106±1). The preservation status of each fetus in the gilt uterus was recorded, and the fetal mortality rate was calculated for each litter. PRRSV RNA concentrations in gilt blood and fetal thymus were determined by quantitative reverse transcription polymerase chain reaction as described previously [5 (link)].
9
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from the samples collected in Tempus™ Blood RNA Tubes using a Tempus™ Spin RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The concentration and purity of the isolated RNA was determined using a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using a High-Capacity cDNA Archive kit (Thermo Fisher Scientific), according to the manufacturer’s instructions, and a peqSTAR thermal cycler (VWR, Radnor, PA, USA).
10
Transcriptome Analysis of Whole Blood
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We made use of pre-processed gene expression data extracted from WPB samples of individuals as provided by the LIFE database. Participant's recruitment, blood collection, storage and mRNA preparation, microarray measurements, and primary data pre-processing was realized by different groups of the LIFE center (Loeffler et al., 2015 (link)). WPB was collected in tempus blood RNA tubes (ThermoFisher, Waltham, MA, USA) and stored at −80°C until further processing. RNA was isolated and then hybridized to Illumina HT-12 v4 Expression BeadChips (Illumina, San Diego, CA, USA) and measured on an Illumina HiScan device. Raw probe level data were extracted using Illumina GenomeStudio and then further pre-processing including batch correction, outlier and missing value removal, log-transformation, quantile normalization, and centralization of the expression value of each gene using an in-house pipeline as described in detail in Supplementary Methods (Supplementary File 1) was undertaken. The final transcriptome data consists of more than 48,000 probe IDs including the expression values of 19,049 genes for each of the individuals.
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