Total RNA was collected using Qiagen Blood RNA kit (Qiagen Inc.) and reverse-transcribed into cDNA using First Strand cDNA synthesis kit (Thermo Fisher Scientific). The primer sequences are given in supplementary table 1 . Quantitative PCR analysis was performed on a QuantStudio 5 Real-Time PCR System (Applied Biosystems) using PowerUP SYBR Green PCR Master Mix (Thermo Fisher Scientific). The PCR primer sequences can be found in the Supplementary Information (supplementary table 1 ). Real Time PCR data analysis was performed by the Livak Method [34 (link)]. Median of fold change of each lncRNA was used for data analysis and clinical correlation.
Blood rna kit
Manufactured by Qiagen
Sourced in Germany
The Blood RNA Kit is a laboratory product designed to extract and purify total RNA from whole blood samples. It provides a standardized and efficient method for isolating high-quality RNA suitable for various downstream applications, such as gene expression analysis and RT-qPCR.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Globinclear kit, by Thermo Fisher Scientific (3 mentions)
Totalprep rna amplification kit, by Illumina (3 mentions)
Iscan machine, by Illumina (2 mentions)
Human ht 12 v4.0 expression beadchips, by Illumina (2 mentions)
Agilent bioanalyser, by Agilent Technologies (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Pcr purification kit, by Qiagen (2 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Stepone plus 96 well system, by Thermo Fisher Scientific (2 mentions)
Hiseq 3000, by Illumina (2 mentions)
Globinclear human kit, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nanodrop 8000, by Agilent Technologies (2 mentions)
Bcl2fastq2 v 2.17 program, by Illumina (2 mentions)
Novaseq, by Illumina (2 mentions)
Powerup sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (1 mentions)
16 protocols using blood rna kit
1
Quantitative PCR Analysis of lncRNAs
2
RNA Isolation and Amplification Protocol
3
IFN-inducible Pharmacodynamics Biomarkers in SSc
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Five type I IFN-inducible genes (RSAD2, IFI44, IFI44L, IFI27, IFI6) were selected as pharmacodynamics markers for MEDI-546 based on their prevalence and magnitude of overexpression in patients with SSc as compared with healthy controls [25 ,26 (link)]. Total RNA was extracted from blood and skin biopsies using the PAXgene Blood RNA kit and the Qiagen RNeasy Fibrous Tissue Mini kit (Hilden, Germany), respectively. RNA purity and concentration were determined spectrophotometrically (260/280 > 1.9). RNA quality was assessed on an Agilent 2100 Bioanalyzer using the RNA 6000 Nano LabChip® (Santa Clara, CA, USA).
The expression level of the 5-gene marker in whole blood and skin was measured using Affymetrix Human Genome U133 Plus arrays (Santa Clara, CA, USA) [25 ]. Blood samples were procured at each evaluation visit, except for days 21 and 22, and skin biopsies were collected on days 0, 7 (single doses only), and 28 (multiple doses only). Transcript profiling was conducted according to standard methods [27 (link)]. The rationale for the definition of positive gene signatures as >2.9 for skin and >1.8 for blood and the details of score generation are included in the Wang publication [26 (link)].
The expression level of the 5-gene marker in whole blood and skin was measured using Affymetrix Human Genome U133 Plus arrays (Santa Clara, CA, USA) [25 ]. Blood samples were procured at each evaluation visit, except for days 21 and 22, and skin biopsies were collected on days 0, 7 (single doses only), and 28 (multiple doses only). Transcript profiling was conducted according to standard methods [27 (link)]. The rationale for the definition of positive gene signatures as >2.9 for skin and >1.8 for blood and the details of score generation are included in the Wang publication [26 (link)].
4
RNA Extraction and RNA-Seq Library Preparation
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RNA was extracted from the PAXgene® Blood RNA tubes using the PAXgene® Blood RNA kit and purified with the RNeasy MinElute Cleanup kit (Qiagen). A total of 90 RNA samples were prepared (30 sheep, 3 time points: T0H, T4H, T24H). The quality of the total RNA was assessed on an Agilent Bioanalyzer 2100, using RNA 6000 pico kit (Agilent Technologies). Directional RNA-Seq Libraries were constructed from 1 µg of total RNA using the TruSeq mRNA Stranded Library Prep Kit (Illumina), following the manufacturer’s instructions. The final libraries’ quality was assessed with an Agilent Bioanalyzer 2100, using an Agilent High Sensitivity DNA Kit. Libraries were pooled in equimolar proportions and sequenced in paired-end runs (51 nt forward–34 nt reverse) on an Illumina NextSeq500 instrument, using NextSeq 500 High Output 75 cycles kits. Demultiplexing has been done with bcl2fastq2 V2.2.18.12. Adapters were trimmed with Cutadapt1.12 and only reads longer than 10 pb were kept.
5
RNA Splicing Analysis of CHD7 Variant
6
Gene Expression Profiling from Blood Samples
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Microarrays for the infant correlates of risk study were performed as previously described72 (link). Briefly, PBMC were thawed and RNA was extracted using an RNeasy kit (Qiagen) according to the manufacturer’s instructions. mRNA was amplified using the Illumina Totalprep kit (Ambion) according to the manufacturer’s instructions, and the quality of the RNA was checked using an Agilent bioanalyser following extraction and again following amplification using the RNA Pico or Nano kits. For whole blood transcriptomic studies, PAXgene® tubes were thawed over two hours at room temperature and total intracellular RNA was extracted using the Blood RNA Kit (Qiagen) according to the manufacturer’s instructions. The purity and quantity of the isolated total RNA was assessed prior to storage at −20 °C until required. Globin mRNA was subsequently depleted using the GLOBINclear Kit (Ambion), amplified and biotin-labelled using the TotalPrep RNA Amplification Kit (Illumina). RNA quality was subsequently assessed. Biotinylated cRNA was hybridised to Illumina HumanHT-12 (v4.0) expression beadchips according to the manufacturer’s instructions. Beadchips were scanned with an Illumina iScan machine, and data extracted using the GenomeStudio software.
7
Gene Expression Analysis of DPP9 in Blood
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Blood RNA Kit (Qiagen, Hilden, Germany) was used to isolate RNA from blood. RNA concentrations were assessed with Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was reversed transcribed to cDNA using High Capacity cDNA Archive Kit (Applied Biosystems, Darmstadt, Germany). Gene expression was assessed by real time PCR using an LightCycler® 480 Real-Time PCR System (Roche Diagnostics SL, Barcelona, Spain) with SYBR green technology suitable for relative genetic expression quantification (Roche Diagnostics SL, Barcelona, Spain). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. The commercially predesigned KiCqStart® primers used were as follows: DPP9 (forward sequence: 5′-CCACCAAGGTTTATCCAATG-3′, reverse sequence: 5′-ACTCATCGACTTCCTCATAC-3′). The optimal sample concentration was 800 nM.
8
Comprehensive RNA Expression Profiling
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Blood samples in PAXgene® tubes were thawed over 2 h at room temperature and total intracellular RNA was extracted using the Blood RNA Kit (Qiagen) according to the manufacturer’s instructions. The purity and quantity of the isolated total RNA was assessed using an Agilent Bioanalyser prior to storage at −20 °C until required. Globin mRNA was subsequently depleted using the GLOBINclear Kit (Ambion). Depleted RNA was then amplified and biotin-labelled using the TotalPrep RNA Amplification Kit (Illumina) and RNA quality assessed using Agilent’s 2100 Bioanalyzer. This was purified and assessed using the Agilent Bioanalyser. Biotinylated cRNA was hybridised to Illumina Human HT-12 v4 Expression Beadchips according to the manufacturer’s instructions. Beadchips were scanned with an Illumina iScan machine, and data extracted using the Illumina’s GenomeStudio 2011 software.
9
Whole Blood Transcriptome Profiling
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Peripheral blood was collected in PAXgene tubes and stored at −80°C until required for whole blood transcriptomic studies as previously described (33 (link)). Samples were subsequently thawed at room temperature for 2 h, total intracellular RNA was extracted using the Blood RNA Kit (Qiagen) and the purity and quantity of RNA was assessed prior to storage at −20°C. Globin mRNA was depleted using the GLOBINclear Kit (Ambion), amplified and biotin-labeled using the TotalPrep RNA Amplification Kit (Illumina). RNA was purified and the quality assessed using an Agilent bioanalyser. Biotinylated cRNA was hybridized to Illumina HumanHT-12 (v4.0) expression beadchips according to the manufacturer's instructions. Beadchips were scanned with an Illumina iScan bead array reader confocal scanner and data extracted using GenomeStudio software.
10
PPMI Early-Stage Parkinson's RNA-Seq Analysis
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Subject recruitment and eligibility criteria for the PPMI study have been previously published66 (link). To increase matching between ICICLE-PD, we limited our analysis to 287 early-stage (diagnosed <13 months) idiopathic (i.e., without a known PD-related genetic diagnosis) PD patients and 176 controls, additionally matching within PPMI for age and sex. Whole blood RNAseq data was downloaded as FASTQ files from the online PPMI repository (https://www.ppmi-info.org/ ). RNA collection, isolation and subsequent sequencing have been previously reported27 (link) and is described in the PPMI Biologicals Manual. Briefly, whole-blood RNA was extracted from PAXgene tubes (Blood RNA Kit, Qiagen), rRNA and globin depleted (Globin-Zero Gold rRNA Removal Kit, Illumina). Library preparation used the NEB/Kapa (NEBKAP) kit (see ref. 27 (link) for more information). Sequencing was performed using an Illumina NovaSeq 6000 generating 125–150 bp paired-end reads. Median sequencing depth was estimated as 107 (IQR = 31.2) million paired-end reads (Supplementary Figure 1 ).
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