Peripheral whole-blood samples were collected via PAXgene RNA tubes (Qiagen, Valencia, CA, USA) and stored at −80 °C until RNA extraction. Total RNA was extracted via the PreAnalytiX PAXgene blood RNA Kit (Qiagen) and automated using the QIAcube System (Qiagen). Quantity and purity of isolated RNA was determined via spectrophotometry (NanoDrop, Thermo Scientific, Waltham, MA, USA). Quality of RNA was confirmed by chip capillary electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA, USA).
Qiacube system
Manufactured by Qiagen
Sourced in Germany, Italy, United States
The QIAcube system is a fully automated sample-to-result platform designed for nucleic acid purification. It automates the complete process from sample preparation to downstream analysis, providing consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp dna mini kit, by Qiagen (11 mentions)
Nanodrop, by Thermo Fisher Scientific (10 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Qiaamp dna blood mini kit, by Qiagen (6 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Taqman advanced mirna cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Paxgene blood mirna kit, by Qiagen (5 mentions)
Qiaamp dna stool mini kit, by Qiagen (4 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Qiaamp viral rna mini kit, by Qiagen (3 mentions)
Qiaexpert quantification system, by Qiagen (3 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Qiaxpert quantification system, by Qiagen (3 mentions)
Nanodrop n1000 system, by Avantor (3 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (3 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (3 mentions)
Paxgene rna tube, by Qiagen (2 mentions)
102 protocols using qiacube system
1
Whole-Blood RNA Extraction and Analysis
2
Semiquantitative RT-PCR Analysis of Splicing Products
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For analysis of splicing products using the pcNLmini-RI vector, semiquantitative RT-PCR analysis was carried out similarly as described previously [29 (link),32 (link)]. Briefly, HEK293T cells were transfected with vectors, and on the next day, the cells were lysed for automatic RNA extraction using a QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany) and QIAcube system (Qiagen). RNA samples were used for cDNA synthesis using an oligo(dT) primer. Semiquantitative PCR reactions were performed using the cDNA samples as templates and specific primer sets for all transcripts, the full and D1/A1 products, and the D1/A1-D2b/A2, D1/A1-D2/A2, and D1/A2 products [29 (link),32 (link)]. PCR amplicons were analyzed by the agarose gel electrophoresis using Metaphor agarose (Lonza Group AG, Basel, Switzerland) followed by visualization using the Amersham Imager 600 instrument (GE Healthcare, Chicago, IL, USA).
3
Evaluating Automated Nucleic Acid Extraction Methods
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Three different automated systems were used for the nucleic acid extraction. QIAcube system (Qiagen, Hilden, Germany) with QIAamp MinElute Virus Spin Kit (Qiagen), EZ1 Advanced XL system (Qiagen) with EZ1 Advanced XL Virus Mini kit v2.0 (Qiagen), and MICROLAB Nimbus IVD system (Hamilton, Reno, NV, USA) with STARMag96 Virus kit (Seegene, Seoul, Korea) were evaluated. Three aliquot samples were separated from each of the specimens, and nucleic acid of each aliquot samples was extracted on the same day by three different automated systems in a single laboratory. The sample and elution volumes used in this study were 150 μL and 60 μL for the QIAcube system, 400 μL and 60 μL for the EZ1 Advanced XL system, and 600 μL and 100 μL for the MICROLAB Nimbus IVD system, respectively. For the quality control of the entire nucleic acid extraction process and RT-PCR, 10 μL of bacteriophage MS2 (AnyplexTMII RV16 detection, Seegene) was added to each sample as the internal control in order to check the entire process from nucleic acid extraction to PCR. cDNA was synthesized using the cDNA Synthesis Premix (Seegene) which included reverse transcriptase and a random hexamer, according to the manufacturer's protocol.
4
Bulk RNA Sequencing of Blood and Fibroblasts
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Sequencing was conducted on blood for 46 patients and cultured fibroblasts for 1 patient due to sample availability. Blood-derived RNA was obtained by collecting peripheral whole blood in PAXgene blood RNA tubes and using the QIAcube system (Qiagen) according to the manufacturer’s protocol for RNA extraction. RNA was isolated from fibroblasts as previously described [34 (link)].
Sequencing libraries were prepared with either the TruSeq RNA Sample Prep Kit v2 or the TruSeq RNA Access Library Prep Kit (Illumina, San Diego, CA). Paired-end 101-basepair reads were sequenced on an Illumina HiSeq 2500 using the TruSeq Rapid SBS sequencing kit version 1 and HCS version 2.0.12.0 data collection software. A median of approximately 200 million reads was generated per individual. Base calling was performed using Illumina’s RTA version 1.17.21.3.
Sequencing libraries were prepared with either the TruSeq RNA Sample Prep Kit v2 or the TruSeq RNA Access Library Prep Kit (Illumina, San Diego, CA). Paired-end 101-basepair reads were sequenced on an Illumina HiSeq 2500 using the TruSeq Rapid SBS sequencing kit version 1 and HCS version 2.0.12.0 data collection software. A median of approximately 200 million reads was generated per individual. Base calling was performed using Illumina’s RTA version 1.17.21.3.
5
Automated DNA Extraction from Whole Blood
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DNA was extracted from whole blood by the DNAeasy Blood&Tissue kit (QIAGEN, Milano, Italy) on a QIAcube System (QIAGEN, Milano, Italy) for automated DNA purification, following the manufacturer’s instructions according to a customized protocol. DNA was quantified and checked for quality using both Quantus™ Fluorometer (Promega, Milano, Italy) and QIAexpert Quantification System (QIAGEN, Milano, Italy). We obtained genetic material suitable for subsequent analytical procedures both from a qualitative (260/280 nm 1.8 for DNA) and quantitative (mean DNA recovery 98.6 ng/µL) point of view.
6
SARS-CoV-2 RNA Extraction from Swabs
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Viral RNA was extracted from 140 µL of nasopharyngeal swab samples using the QIAmp viral RNA mini kit (QIAGEN, Hilden, Germany) and the automatized QIAcube system (QIAGEN, Hilden, Germany), and was eluted in 50 µL of buffer following the manufacturer’s instructions. The positive (EDX SARS-CoV-2 Standard, Exact Diagnostics, Forth Worth, TX, USA) and negative (EDX SARS-CoV-2 Negative, Exact Diagnostics, Forth Worth, TX, USA) controls were also extracted using the same procedure.
7
RNA Extraction and Microarray Analysis of Bronchial Epithelial Cells
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mRNA was extracted from bronchial epithelial cells suspended in Trizol solution using the QIACube system (QIAGEN INC, Valencia, CA). Microarray experiments were performed as we previously reported.50 (link) RNA quality was determined using the Agilent Bioanalyzer 2100 (Agilent Technologies Inc, Santa Clara, CA). RNA was hybridized to Agilent Human GE 4×44K V2 Gene Expression microarrays. The microarrays were scanned using the Agilent Microarray Scanner and the data was extracted using the Agilent Feature Extraction software 10.7.3.1. The data was then normalized using a cyclic loess algorithm authored in the R programming environment using the Bioconductor suite of tools. The resultant data was analyzed using BRB ArrayTools version 4.3.0 developed by Dr. Richard Simon and the BRB-ArrayTools Development Team. Differentially expressed genes were determined by t-test using a false discovery rate of 0.05 with a confidence interval of 90%. The data discussed in this manuscript have been deposited in NCBI’s Gene Expression Omnibus database (GEO, http://www.ncbi.nlm.nih.gov/geo ) and are accessible through GEO Series accession number GSE43696.
8
Fecal DNA Extraction and 16S rRNA Sequencing
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DNA extraction was performed using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) with the following modifications22 (link). Briefly, 250 µL of the fecal sample was placed into a 2-mL tube containing 0.3 g sterile 0.1 mm zirconia beads (BioSpec, OK, USA) with 1.2 mL ASL lysis buffer from QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) and vortexed for 2 min. The samples were subsequently heated at 95 °C for 15 min and then subjected to two cycles of bead beating at a frequency of 30 Hz for 1 min using the Qiagen TissueLyser II. After centrifugation, 1.2 mL of the supernatant was treated with an InhibitEX Tablet, and 350 µL of the resulting supernatant was used in the subsequent steps with a QIAcube system (Qiagen, Hilden, Germany). Total DNA was eluted in 200 μL of elution buffer and stored at −20 °C until use.
Library preparation of the V3–V4 hypervariable region of the 16S rRNA gene was performed according to the 16S Metagenomic Sequencing Library Preparation Illumina protocol (Part #15044223 Rev. B; Illumina, San Diego, CA, USA). The library pool containing equal molar quantities of each sample was sequenced using the MiSeq. 2 × 300 system (Illumina).
Library preparation of the V3–V4 hypervariable region of the 16S rRNA gene was performed according to the 16S Metagenomic Sequencing Library Preparation Illumina protocol (Part #15044223 Rev. B; Illumina, San Diego, CA, USA). The library pool containing equal molar quantities of each sample was sequenced using the MiSeq. 2 × 300 system (Illumina).
9
DNA Extraction from Whole Blood and Renal Biopsies
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DNA extraction was performed on all samples of whole blood and renal biopsies using an automated QIAcube System according to the DNAeasy Blood and Tissue kit procedure (Qiagen, Milano, Italy) as previously described (10 (link)). Details of DNAmAge analysis are reported in the Supplementary Materials .
10
Quantitative RNA Expression Analysis
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RNA isolation and qPCR analysis have previously been described [38 (link)]. RNA was isolated from approximately 20mg of pulverized tissue by using the QIAcube system (Qiagen, Valencia, CA, USA) following the manufacturer’s protocols. The quantity and quality of RNA were determined on a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). RNA (1 μg) was converted to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystem, Inc., Carlsbad, CA, USA) following the manufacturer’s protocols. Real time PCR reactions contained the following: 1X SYBR Green Master Mix (Applied Biosystem, Inc., Carlsbad, CA, USA), 300 nM of forward and reverse primers, and 40 ng cDNA. Real Time PCR was performed on a Quantstudio 12k flex Real-Time PCR System (Applied Biosystem, Inc., Carlsbad, CA, USA). Real-time qPCR primers were designed and purchased from IDT (Coralville, IA, USA). Expression of candidate genes was normalized to β-actin expression levels and evaluated in comparison to the control (SO diet) using the ΔΔCT method. Primer pairs for gene expression are presented in Table A2 .
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