Total RNA was extracted from the frozen blood samples using a PAXgene Blood RNA Isolation Kit (QIAGEN) according to the manufacturer’s protocol. RNA quality was verified on an Agilent 2100 Bioanalyzer and the RNA quantity was determined using a NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Genes were selected based on higher statistical significance, greater absolute fold-changes, and clinical significance and were tested by qRT-PCR to confirm the results of RNA-Seq analysis. DEFA3 (defensin alpha 3), JUN (jun proto-oncogene), ELANE (neutrophil elastase), and CD177 (CD177 molecule) were selected for validation experiments between the CI and control groups, whereas CEACAM8 (CEA cell adhesion molecule 8), CRISP3 (cysteine-rich secretory protein 3), DEFA3, and RNASE3 (ribonuclease A family member 3) were selected to confirm the differential expression between the CI-preterm and CI-term groups. Four micrograms of RNA was used for cDNA synthesis with a Maxime RT PreMix Kit (iNtRON Biotechnology, Seoul, South Korea). PCRs were performed using 2× Rotor-Gene SYBR Green PCR Master Mix (QIAGEN) in the Rotor-Gene Q (QIAGEN). Primer sequences are shown in Supplemental Table 1 . Actin was used as the housekeeping control. Each sample was run in triplicate, and all samples had standard deviation < 0.5 between the triplicates.
Paxgene blood rna isolation kit
Manufactured by Qiagen
Sourced in United States
The PAXgene Blood RNA Isolation Kit is a laboratory equipment product designed to extract and purify RNA from whole blood samples. It utilizes a proprietary technology to stabilize and preserve the RNA content of the samples, enabling effective RNA isolation and extraction.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Rotor gene sybr green pcr master mix, by Qiagen (1 mentions)
Rotor gene q, by Qiagen (1 mentions)
Maxime rt premix kit, by iNtRON Biotechnology (1 mentions)
Affymetrix human genome u133 plus 2.0 array, by Thermo Fisher Scientific (1 mentions)
Paxgene blood tubes, by Qiagen (1 mentions)
Humanht 12 v3.0 expression beadchip, by Illumina (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 nano labchip kit, by Agilent Technologies (1 mentions)
Sta compact analyzer, by Diagnostica Stago (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Iscan confocal scanner, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Superprep cell lysis rt kit for qpcr, by Toyobo (1 mentions)
Goscrip reverse transcriptase, by Promega (1 mentions)
Thunderbird probe qpcr mix, by Toyobo (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
9 protocols using paxgene blood rna isolation kit
1
Whole Blood RNA Extraction and Gene Expression Analysis
2
Transcriptomic Profiling of Total RNA
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A full description has been provided in our primary analysis study reported previously (30 (link)). In brief, total RNA was isolated using PAXgene Blood RNA Isolation Kit (Qiagen, Valencia, CA, USA) and stored at −80°C. Transcriptomic profiling was performed by hybridizing each RNA sample to Affymetrix Human Genome U-133 Plus 2.0 arrays (Affymetrix Inc., Santa Clara, CA, USA) by Expression Analysis Inc. (Durham, NC, USA) according to the manufacturer’s protocol. Each array contains 54,675 25-mer probe sets that include approximately 47,000 transcripts and variants out of which 38,500 are well-characterized human genes (33 (link)). Gene expression data were then submitted to Information Management Consultants (IMC), Inc. (Reston, VA, USA) for data warehousing using IMC’s Pharmacogenomics Knowledge Management System.
3
Dose-Dependent Immune Response to LPS
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Caucasian male volunteers were given a single bolus of 1 ng/kg (n = 7), 2 ng/kg (n = 6), or 4 ng/kg (n = 7) E. coli LPS (US standard reference endotoxin, kindly provided by Anthony Suffredini, National Institute of Health, Bethesda, MD) and monitored for the duration of 24 h (Supplemental Table 1 ). Blood was collected before (n = 20) and 4 h after LPS administration in PAXgene blood tubes (Qiagen, Venlo, the Netherlands). Total RNA was isolated by means of the PAXgene blood RNA isolation kit (Qiagen) according to the manufacturer's instructions. Total RNA (RNA integrity number (RIN) > 6.0) was processed and hybridized to microarray chips according to Affymetrix or Illumina specifications. The 1 ng/kg (accession: GSE36177)9 and 4 ng/kg (accession: GSE48119)14 samples were hybridized to the Illumina HumanHT‐12 V3.0 expression beadchip. The 2 ng/kg LPS samples (accession: GSE108685)13 were hybridized to Affymetrix human genome U219 96‐array chips. Written informed consent was obtained from all subjects. All studies, data, and ethical statements were collected and handled in accordance with institutional guidelines and ethics committees.
4
Blood RNA Isolation and Quantification
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Blood samples were collected from the patients using PAXgene Vacutainer tubes (BD Biosciences, Franklin Lakes, NJ, USA) and subjected to RNA isolation and extraction using a PAXgene Blood RNA Isolation kit (Qiagen, Inc., Valencia, CA, USA), following the manufacturer's instructions. The quantity of total RNA obtained was determined using a NanoDrop Spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the 280/260 nm absorbance ratio, and RNA integrity was evaluated using an RNA 6000 Nano LabChip kit (Agilent Technologies, Inc., Santa Clara, CA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.).
5
Genome-wide Expression Profiling of Blood
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Blood samples were collected at approximately the same time of day for each participant for all three time points to control for potential circadian rhythm effects on gene expression. Total RNA was extracted using the PAXgene™ Blood RNA Isolation Kit (Qiagen, Valencia, CA, USA). Genome-wide expression of each sample was assessed with a Human Genome U-133 Plus 2.0 array (Affymetrix Inc., Santa Clara, CA, USA) by Expression Analysis Inc. (Durham, NC). Briefly, the double-stranded cDNA was used in a T7 RNA polymerase in vitro transcription reaction (Ambion, Austin, TX, USA) containing biotin-labeled ribonucleotides CTP and UTP. The resulting labeled cRNAs were then hybridized to HG-U133plus2.0 arrays.
6
Comprehensive Thrombophilia Screening Protocol
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For each patient (median age, 31.5 years), the clinical profile and laboratory investigations (hemogram, PT, aPTT, Fibrinogen, D-dimer) were performed along with the complete thrombophilia screening including Protein C and S deficiency and Antithrombin-III deficiency. Protein C and Protein S activities were determined by using commercially available STACLOT kit on an automated STA compact analyzer (Diagnostica Stago). The quantification of AT III was done using STACHROM AT III kit in STA analyzer. Activated protein C resistance (APCR) was carried out using the Stago kit. PCR-RFLP method was used to perform mutational analysis for Factor V Leiden (1691G/A, rs6025), prothrombin (20210G/A, rs1799963), tissue factor plasmin inhibitor (-536C/T), fibrinogen-β (148C/T), MTHFR (677C/T) and plasminogen activator inhibitor-1 (16754G/5G) were performed by PCR-RFLP method. Total RNA from whole blood and plasma were isolated using PAXgene Blood RNA Isolation Kit (Qiagen) and TRIZOL reagent (Sigma) respectively, according to the manufacturer's instructions. miRNA expression and mRNA expression were analyzed as described earlier. Microparticle-TF activity assay was performed in human plasma using MP-TF assay kit (Aniara) according to the manufacturer's instructions.
7
Transcriptome Profiling of Blood and Nasal Swabs
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Total RNAs from whole blood and NP swabs were extracted using the PAXgene blood RNA isolation kit (PreAnalytiX) and the RNeasy Mini kit (QIAGEN, Hilden, Germany), respectively. Biotinylated amplified complementary RNA (cRNA) was generated by in vitro transcription of total RNA using Ambion Illumina TotalPrepRNA amplification kit (Ambion Inc., Austin, TX), according to the manufacturer’s instructions. After purification, 800 ng of cRNA from NP samples and 2000-ng labeled cRNA from blood were hybridized to the HumanHT-12 v4 BeadChip array at 55°C for 18 hours and under washing, blocking, and streptavidine-Cy3 staining steps, according to the manufacturer’s instructions. Each array on the HumanHT-12 v4 Expression BeadChip targets more than 31 000 annotated genes with 47 231 probes derived from the National Center for Biotechnology Information Reference Sequence RefSeq Release 38 (November 7, 2009) and other sources. The array was scanned using an iScan confocal scanner (Illumina Inc., San Diego, CA).
8
Gene Expression Analysis Protocol
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Total RNA isolation and reverse transcription were performed from cells using SuperPrep Cell Lysis & RT Kit for qPCR (TOYOBO, Tokyo, Japan) and from peripheral whole bloods using PAXgene Blood RNA Isolation Kit (QIAGEN, Venlo, The Netherlands) and GoScrip Reverse Transcriptase (Promega, Madison, WI) following the manufacturers’ protocols. The following primers for genes were used: ATP6AP2 [(P)RR; forward 5′-AGG CAG TGT CAT TTC GTA CC-3′ , reverse 5′-GCC TTC CCT ACC ATA TAC ACT C-3′ ], TNFA (TNF-α; forward 5′-ACT TTG GAG TGA TCG GCC-3′ , reverse 5′-GCT TGA GGG TTT GCT ACA AC-3′ ), CFD (forward 5’-GAC ACC ATC GAC CAC GAC-3′ , reverse 5′-GTT GAC TAT GCC CCA GCC-3′), LRG1 (forward 5′-GTT GGA GAC CTT GCC ACC T-3′ , reverse 5′-GCT TGT TGC CGT TCA GGA-3′ ), ACTB (β-actin; forward 5′-CTG GAA CGG TGA AGG TGA CA-3′, reverse 5′-AAG GGA CTT CCT GTA ACA ATG CA-3′) , and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; forward 5′-AGG TCG GTG TGA ACG GAT TTG-3′, reverse 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′ ). TaqMan probes for REN (prorenin) were purchased from Thermo Fisher Scientific. Real-time qPCR was performed using the GoTaq qPCR Master mix (Promega), THUNDERBIRD Probe qPCR Mix (TOYOBO), and StepOne plus Systems (Thermo Fisher Scientific).
9
Cardiovascular Disease miRNA Analysis
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Whole peripheral blood samples (2 mL) were washed and processed for total RNA extraction using the PAXgene Blood RNA Isolation kit (Qiagen, Valencia, CA, USA). Samples were stored at −70 °C until PCR was performed. Prior to PCR, the quality of RNA was documented for quality and fidelity. Total RNA was then diluted 1:10, then 5 μL was reverse-transcribed using the TaqMan microRNA Reverse Transcription Kit (ABI) to create cDNA. Qiagen Human Cardiovascular Disease miRNA PCR Array MIHS-113Z was used with 3 μL aliquots of cDNA, miScript Universal Primer, and SYBR Green PCR master mix in a real-time PCR cycler.
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