Two blood samples per patient were drawn from the cubital vein and collected into PAXgene tubes (BD Biosciences, Franklin Lakes, NJ, USA) in the morning between 8 and 10 a.m, the first one at the hospital arrival and the second one at the hospital discharge. Samples were incubated 2 h at room temperature to ensure complete lysis of blood cells. PAXgene tubes were stored in the freezer (−80 °C) following commercial specifications (Qiagen, Hilden, Germany). The tubes were first transferred to −20 °C (24–72 h) and then transferred to −80 °C freezer until further processing.
Paxgene tube
Manufactured by BD
Sourced in United States
PAXgene tubes are a type of blood collection tubes used for the stabilization and preservation of RNA in whole blood samples. They are designed to facilitate the collection, transport, and storage of blood samples for downstream RNA analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Paxgene blood rna kit, by Qiagen (8 mentions)
2100 bioanalyzer, by Agilent Technologies (5 mentions)
Paxgene blood mirna kit, by Qiagen (4 mentions)
Novaseq 6000, by Illumina (4 mentions)
Paxgene tube, by Qiagen (2 mentions)
Paxgene blood rna kit, by BD (2 mentions)
Paxgene blood rna kit, by Preanalytix (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Edta tube, by Sarstedt (2 mentions)
Humanht 12 v4 expression beadchip, by Illumina (2 mentions)
Totalprep rna, by Illumina (2 mentions)
Ficoll paque plus reagent, by GE Healthcare (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green supermix, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Globinclear kit, by Thermo Fisher Scientific (1 mentions)
Globinclear, by Thermo Fisher Scientific (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
On column dnase digestion, by Qiagen (1 mentions)
44 protocols using paxgene tube
1
Blood Sampling and Storage Protocol
2
Blood RNA Extraction and qPCR Analysis
3
Corpus Luteum Dynamics and Pregnancy Evaluation in Dairy Cows
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Blood samples were collected immediately before AI (day 0) and on days 7 and 15 of the experiment from either the coccygeal vein or artery into blood collection tubes. Transrectal ultrasonography (7.5-MHz transducer; 500 V, Aloka, Wallingford, CT) was performed concurrently with blood sampling on days 0, 7, and 15 to verify dominant follicle diameter (day 0) and estimate corpus luteum (CL ) volume (days 7 and 15). After ultrasonography on day 15, cows diagnosed without the presence of a CL on day 0, but with a CL greater than 0.38 cm3 in volume on days 7 and 15 (two or three cows per pen; CSSO, n = 20; CON, n = 24), were assigned to conceptus collection via transcervical flushing followed by endometrial biopsy in the uterine horn ipsilateral to the CL (Cipriano et al., 2016 (link)). All cows returned to their original pens after conceptus collection and endometrial biopsy. On day 20, blood samples were collected from the nonflushed cows (two or three cows per pen; CSSO, n = 25; CON, n = 21) into PAXgene tubes (BD Diagnostics, Sparks, MD) for whole-blood RNA extraction. On day 21, treatment administration was terminated, and blood samples were collected from the nonflushed cows on day 30 for pregnancy evaluation.
4
RNA-Seq Protocol for Transcriptomic Analysis
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Briefly, peripheral blood was drawn by venipuncture into PAXgene tubes (BD Diagnostics), and RNA was isolated using standard protocols (Qiagen, Inc.). Globin transcripts were removed using GLOBINclear™ kit (Life Technologies), and samples were prepared for sequencing using the Encore complete kit (NuGEN). Paired-end sequencing of 100-bp reads was performed using the Illumina HiSeq 2000 by standard procedures. The quality of sequencing data was checked using the FastQC v0.11.2 tool (Supplementary Table 7 ); sequencing adapters were removed using Trimmomatic v0.33.19 (link) Short reads were aligned to the GRCh38/hg38 human genome assembly using TopHat v2.0.9.20 (link) A matrix of raw counts per gene was assembled using htseq-count21 (link) and Homo_sapiens.GRCh38.80.gtf gene models obtained from the Ensembl web site. A matrix of quartile-normalized fragments per kilobase of exons per million reads mapped (FPKM) counts was calculated using the cuffnorm v2.2.1 tool.20 (link) To filter out low expressed signals, as recommended by several method comparison studies,14 (link),22 (link)–24 (link) genes having zero FPKM expression in at least one sample were removed, keeping 18,697 expressed genes out of 65,217 total gene models.
5
Whole Blood RNA Extraction and Analysis
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Whole blood was obtained by venipuncture into PAXGene tubes (BD company) and RNA was extracted with on-column DNase digestion (Qiagen). Excess globin transcripts were removed using GLOBINclear (Ambion). RNA concentrations were determined using a NanoDrop spectrophotometer and RNA quality was assessed using the RNA 6000 Nano kit on the Bioanalyzer 2100 (Agilent) with quality threshold RIN scores > 8.
6
RNA Isolation from Whole Blood MPN
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Whole blood from patients with MPN and healthy donors was collected into PAXgene tubes (BD Biosciences) containing red blood cell lysis buffer. RNA was isolated using PAXgene Blood RNA kit, following the manufacturer’s instructions (762164, PreAnalytiX, Hombrechtikon, Switzerland).
7
Whole Blood RNA Isolation from MPN Patients
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Whole blood from MPN patients and healthy donors was collected into PAXgene tubes (BD Biosciences) containing RBC lysis buffer . RNA was isolated using PAXgene Blood RNA kit following manufacturer instructions (762164, PreAnalytix, Switzerland).
8
Standardized Blood Collection for Multispecies RNA Analysis
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Blood samples were collected in triplicates from six rabbits, six mice, six human donors and four NHP. Blood was collected into PAXgene tubes (#BD762165, BD Biosciences) for mouse, NHP and human samples or into lithium heparin tubes for the rabbit samples (#13,526,530, Greiner). For PAXgene conservation, a blood:reagent ratio of 2.5:6.9 was conserved, regardless the species [14 (link)–17 (link)]. Next, 150 µl (mouse), 250 µl (NHP) and 2.5 ml (human) of each blood sample was dispensed into 1.5 ml Eppendorf tubes or 15 ml Falcon tubes to which 414 µl, 690 µl and 6.9 ml PAXgene reagent were added, respectively. For the rabbit samples, 3 ml of total blood were dispensed into 3 ml lithium-heparin tubes (#13,526,530, Greiner). 1 ml of heparinized blood was then mixed with 2.8 ml of PAXgene reagent into 15 ml Falcon tube (#352,097, Falcon). After collection, the tubes were inverted 10 times and stored upright at room temperature (18 °C–25ºC) for a minimum of 2 h and a maximum of 72 h before transferring to a freezer at -20 °C for 24 h and then at -80 °C until RNA extraction.
9
Transcriptional Profiling of Peripheral Blood
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Peripheral whole blood was collected in PAXgene tubes (BD Biosciences, San José, CA, USA) at baseline, day 85, year 1, and year 2 for transcriptional profiling. RNA was isolated from all available samples (Qiagen Kit, Hilden, Germany) following the manufacturer’s instructions.
Transcriptional profiling was performed using Affymetrix U219 GeneChips. Blood samples were collected over a 3-year period (2009–2012). Samples were processed in two batches: an early cohort comprising baseline and day 85 and a late cohort comprising year 1 and year 2 samples. The strategy to remove batch effect was as follows: RNA profiling from a representative set of 54 patients (9.5% of the 568 patients at baseline) was repeated in the late cohort; a linear model was used to compute the batch-specific differences using the repeated samples. Poor probes and non-informative gene matches were sequentially eliminated via an alternative chip definition file (BrainArray Ensembl chip definition file based on Human Genome build GRCh37) [21 ] and I/NI filter, respectively.
Transcriptional profiling was performed using Affymetrix U219 GeneChips. Blood samples were collected over a 3-year period (2009–2012). Samples were processed in two batches: an early cohort comprising baseline and day 85 and a late cohort comprising year 1 and year 2 samples. The strategy to remove batch effect was as follows: RNA profiling from a representative set of 54 patients (9.5% of the 568 patients at baseline) was repeated in the late cohort; a linear model was used to compute the batch-specific differences using the repeated samples. Poor probes and non-informative gene matches were sequentially eliminated via an alternative chip definition file (BrainArray Ensembl chip definition file based on Human Genome build GRCh37) [21 ] and I/NI filter, respectively.
10
Transcription Factor Expression Profiling
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Whole blood was obtained by venous puncture and stored in PAXgene tubes (BD Biosciences) at -80°C, for a period below six months. Whole RNA was prepared using PAXgene Blood RNA Kit (Qiagen) according to the manufacturer’s instructions. RNA was quantified on a Nanodrop ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA). cDNA synthesis carried out using the Superscript III RT-PCR kit (Applied Biosystems, Branchbug, NJ, USA). Taqman real time PCRs were performed via the universal PCR Master Mix (x2) and specific primers and probes (Applied Biosystems). Briefly, PCR was performed in the ABI Prism 7000 sequence detection system (Applied Biosystems) at 50°C for 2 min, 95°C for 10 min, 45 cycles of 95°C for 15 s, and 60°C for 1 min. The studied genes were TBX21 (Hs00203436), GATA3 (Hs00203436), RORC (Hs01076122), STAT3 (Hs00374280), STAT4 (Hs00374280), STAT6 (Hs00598625) and FOXP3 (Hs01085834). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 5’-CCGCATCTTCTTGTGCAGTG-3’) was used as an endogenous control and mRNA were quantified using the ΔCt method [ΔCt = Ct (target gene)—Ct (endogenous gene)]. qPCR conditions were the same as described above for the gene expression analysis (Applied Biosystems).
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