Blood from GRACE participants had been stored in PAXgene tubes and frozen at -80°. After thawing, RNA was extracted from whole blood using the Qiagen PAXgene Blood RNA System [10 (link)]. Quantification was performed using Qubit RNA HS Assay (Thermo Fisher Scientific) and analysis of RNA integrity was performed using Fragment Analyzer DNF-472 High Sensitivity RNA (Advanced Analytical). RNA was normalized to 2ng/uL or less in 5uL based on Qubit values. External RNA Controls Consortium (ERCC) controls (Thermo Fisher Scientific) were spiked into the samples by adding 1μL at 25pg/μL as quantified by Qubit. Reverse transcription and library preparation were performed using New England Biolab’s NEBNext Ultra II RNA Library Prep (E7770), with an RNA fragmentation time of 8 minutes and 18 cycles of indexing PCR using custom TruSeq indexing primers. Samples were prepared in two batches using the Agilent Bravo Automated Liquid Handling Platform. A water control was included in each batch. For each ten of the RNA samples, a technical replicate was included. Prior to deep sequencing on a NovaSeq (Illumina), relative library concentrations of each sample were determined by pooling 1μL of each and sequencing on a MiSeq (Illumina) with an average of 360,000 read pairs/sample (Fig 1A ).
Paxgene blood rna system
Manufactured by Qiagen
Sourced in Germany
The PAXgene Blood RNA System is a laboratory equipment product that is designed to collect, stabilize, and store blood samples for the purpose of RNA analysis. It provides a standardized and consistent method for preserving the RNA content of blood samples, which is crucial for accurate and reliable gene expression studies.
Automatically generated - may contain errors
Lab products found in correlation
Truseq stranded mrna library prep kit, by Illumina (2 mentions)
Agilent 4200 tapestation, by Agilent Technologies (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Agilent bravo automated liquid handling platform, by Agilent Technologies (1 mentions)
Nebnext ultra 2 rna library prep, by New England Biolabs (1 mentions)
Novaseq, by Illumina (1 mentions)
Qubit rna hs assay, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Viiatm 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 instrument, by Illumina (1 mentions)
Paxgene rna tube, by Qiagen (1 mentions)
1 kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
Generead rrna depletion kit, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Experion automated electrophoresis system, by Bio-Rad (1 mentions)
Paxgene rna stabilization tubes, by Qiagen (1 mentions)
Primer express software, by Thermo Fisher Scientific (1 mentions)
16 protocols using paxgene blood rna system
1
Comprehensive RNA sequencing protocol
2
RNA Extraction and qRT-PCR Analysis
3
Robust RNA Extraction and Sequencing
4
Whole Blood RNA Extraction for mRNA-seq
5
Analyzing CENPJ Missense Variant's Impact on Splicing
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To explore the possible consequences of the CENPJ missense variant on splicing accuracy, RNA was extracted from the patient’s blood using the PAXgene blood RNA system (QIAGEN, Hilden, Germany) and subsequently converted to cDNA as described previously [22 (link)]. In brief, RT-PCR was performed using forward 5′-AGCCACTTGAACCACTGAAC-3′ and reverse 5′-CAGTCTGGTCAGGAAACGTG-3′ primers to amplify the relevant portion of CENPJ. Amplicons were resolved on a 2% agarose gel along with a 1 kb plus DNA ladder (10787018, Thermo Fisher Scientific, Waltham, MA, USA) as the size marker. Each band was Sanger sequenced separately to determine the effects on splicing.
6
Comprehensive RNA Extraction and Purification
7
RNA Extraction and cDNA Synthesis Protocol
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Blood samples (2.5ml) were collected using PAXgene tubes. RNA isolation protocol followed the PAXgene blood RNA system (QIAGEN, Australia). RNA extraction protocol, as per manufacturer’s instructions, was followed. RNA samples from tissues were extracted using the RNeasy plus mini kit (QIAGEN, Australia). The yield of total RNA and the 260/280 ratio was measured using the Nanodrop® ND-1000 spectrophotometer (NanoDrop Technologies, USA). cDNA was synthesised from 5μg of total RNA using random decamer primers (Geneworks, Australia) and AffinityScriptTM multiple temperature reverse transcriptase (Stratagene, USA).
8
Whole Blood RNA Stabilization and Purification
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We collected peripheral whole blood samples (2.5 mL) in PAXgene RNA stabilization tubes (QIAGEN Inc., Valencia, CA, U.S.A.) at each visit of those enrolled for illness and froze the samples at—80°C until RNA purification to minimize gene expression changes induced by handling and processing. RNA purification was performed using the PAXgene Blood RNA system (QIAGEN Inc., Valencia, CA) according to manufacturer’s instructions. Quality control of RNA samples was performed using spectrophotometry (NanoDrop-1000 Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, U.S.A.) and microfluidic electrophoresis (Experion Automated Electrophoresis System, Bio-Rad Laboratories, Hercules, CA).
9
Quantitative PCR Analysis of Gene Expression
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Blood was collected and prepared as described using the PAXgene Blood RNA system (Qiagen,Valencia, CA, USA)[28] (link). Samples with RNA integrity values > 7.0 and ratio of absorbances at 260/280 nm between 1.7 and 2.4 were used in the current study. Primer Express software (Life Technologies, Carlsbad, CA, USA) was used to design the primers. The High Capacity RNA transcription kit (Life Technologies, Carlsbad, CA, USA) was used to reverse transcribe 1 µg of total RNA according to the manufacturer's protocol. The DNA engine Opticon 2 Analyzer (Bio-Rad Life Sciences, Hercules, CA, USA) was used for the qPCR reactions. Each 25 µl reaction contained Power SYBR (Life Technologies, Carlsbad, CA, USA) and primers at a concentration of 5 µM. Primer sequences used in qPCR assays are as follows: GAPDH; forward: 5′- CAACGGATTTGGTCGTATTGG-3′ ; reverse: 5′- TGATGGCAACAATATCCACTTTACC-3′ , SOD2; forward: 5′- GTTCAATGGTGGTGGTCATATCA-3′ ; reverse: 5′- GCAACTCCCCTTTGGGTTCT-3′ . Amplification conditions and detailed description of qPCR experiments is described in [15] (link).
10
RNA-Seq Analysis of Cancer Transcripts
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RNA extraction from PBMCs was performed following the instructions of the RNeasy RNA blood mini kit (QIAGEN). RNA from PAXgene blood tubes was extracted using the PAXgene Blood RNA System (Qiagen) following the manufacturer’s protocol. RNA abundance was measured photometrically (Xpose), and RNA integrity number (RIN) was determined (Agilent Bioanalyzer).
Cancer-related transcripts were enriched and sequenced according to the method described in detail in ref 25 . Briefly, 50 ng total RNA were reversely transcribed with oligo-dT using kit SQK-PCB109 from Oxford Nanopore Technologies and amplified by PCR. cDNAs were captured with a custom Agilent SureSelect probe set directed against 123 hereditary cancer genes. Following a second PCR amplification of captured cDNAs, sequencing libraries were prepared with the SQK-PCB109 kit and sequenced on R9 flow cells on the GridION. Capture and sequencing was performed in triplicates. Differential expression analysis has been performed against 21 reference samples in triplicates using the Oxford Nanopore Technologies Pipeline for differential gene expression (DGE) and differential transcript usage analysis of long reads (https://github.com/nanoporetech/pipeline-transcriptome-de ), which uses minimap2 V.2.18,21 (link) salmon V.1.5.026 (link) and edgeR V.3.34.0.27 (link)
Cancer-related transcripts were enriched and sequenced according to the method described in detail in ref 25 . Briefly, 50 ng total RNA were reversely transcribed with oligo-dT using kit SQK-PCB109 from Oxford Nanopore Technologies and amplified by PCR. cDNAs were captured with a custom Agilent SureSelect probe set directed against 123 hereditary cancer genes. Following a second PCR amplification of captured cDNAs, sequencing libraries were prepared with the SQK-PCB109 kit and sequenced on R9 flow cells on the GridION. Capture and sequencing was performed in triplicates. Differential expression analysis has been performed against 21 reference samples in triplicates using the Oxford Nanopore Technologies Pipeline for differential gene expression (DGE) and differential transcript usage analysis of long reads (
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