Islet RNA was extracted either with the QIAGEN RNeasy Mini kit (#74106, QIAGEN, Valencia, California, USA) or TRI Reagent solution (#T9424, Sigma-Aldrich, Sydney, New South Wales, Australia), according to the manufacturer’s instructions. cDNA was generated from 500 ng of RNA and random hexamer primers using the Maxima First Strand cDNA Synthesis kit for RT-qPCR (#K1641, ThermoFisher Scientific, Scoresby, Victoria, Australia). Real-time PCR was performed in ABI Prism 7900HT Sequence Detection System (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) using specific primers (mouse sequences in Table S2 and human sequences in Table S3 ) and Power SYBR Green mastermix (#4367659, Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) as previously described (Lalwani et al., 2014 (link)). Differences in gene expression were calculated using the ΔΔCT method. Primers for enterovirus (EV) mRNA targeted the highly conserved 5′ untranslated region (UTR) of the EV genome as previously described (Craig et al., 2003 (link)). Therefore, this measures viral load rather than individual translated viral RNAs. For many of the gene expression changes after viral infection, fold changes were very large (> 10-fold) and for those genes, data were log10 transformed for statistical analysis.
Tri reagent solution
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
TRI Reagent solution is a versatile reagent used for the isolation of high-quality RNA, DNA, and protein from a variety of biological samples. It is a single-step liquid reagent designed to facilitate the extraction and purification of these biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (7 mentions)
Nanodrop, by Thermo Fisher Scientific (6 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Abi 7900t pcr system, by Thermo Fisher Scientific (3 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Maxima first strand cdna synthesis kit for rt qpcr, by Thermo Fisher Scientific (2 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (2 mentions)
Itaq universal sybr green supermix, by Bio-Rad (2 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Transcription first strand cdna synthesis kit, by Roche (2 mentions)
Dnase treatment, by Qiagen (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Cfx touch real time pcr detection system, by Bio-Rad (2 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
High capacity cdna kit, by Thermo Fisher Scientific (2 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (2 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rotor gene 6000, by Qiagen (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
49 protocols using tri reagent solution
1
RNA Extraction and RT-qPCR Analysis of Islet Samples
2
RNA Extraction and RT-qPCR Analysis of Islet Samples
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Islet RNA was extracted either with the QIAGEN RNeasy Mini kit (#74106, QIAGEN, Valencia, California, USA) or TRI Reagent solution (#T9424, Sigma-Aldrich, Sydney, New South Wales, Australia), according to the manufacturer’s instructions. cDNA was generated from 500 ng of RNA and random hexamer primers using the Maxima First Strand cDNA Synthesis kit for RT-qPCR (#K1641, ThermoFisher Scientific, Scoresby, Victoria, Australia). Real-time PCR was performed in ABI Prism 7900HT Sequence Detection System (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) using specific primers (mouse sequences in Table S2 and human sequences in Table S3 ) and Power SYBR Green mastermix (#4367659, Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) as previously described (Lalwani et al., 2014 (link)). Differences in gene expression were calculated using the ΔΔCT method. Primers for enterovirus (EV) mRNA targeted the highly conserved 5′ untranslated region (UTR) of the EV genome as previously described (Craig et al., 2003 (link)). Therefore, this measures viral load rather than individual translated viral RNAs. For many of the gene expression changes after viral infection, fold changes were very large (> 10-fold) and for those genes, data were log10 transformed for statistical analysis.
3
Quantitative PCR Assay for Stem Cell Markers
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Total RNA was isolated from cells using TRI-reagent solution (Sigma) and treated with DNase (Invitrogen, ThermoFisher Scientific). Reverse transcription was performed using random hexanucleotides as primers and highscript reverse transcriptase (Bio-Rad, Hercules, CA, USA) according to standard procedures. Quantitative (q) PCR was carried out using the power SYBR Green PCR Master Mix (Bio-Rad), according to manufacturer’s instructions. Primers specific for rat G6pd were used for normalization of the data. Primer sequences were as follows: rat Pou5f1 (5′-CCTGCAGCAGATCACTAGCAT-3′ / 5′-CACTCGAACCACATCCCTCT-3′); rat Sox2 (5′-ACCGTGATGCCGACTAGAAA-3′ / 5′-GCGCCTAACGTACCACTAGAA-3′); rat Nanog (5′-GTCTGCTACTGAGATGCTCT-3′ / 5′-ATCTGCTGGAGGCTGAGGTA-3′); rat G6pd (5′-TCCTCTATGTGGAGAATGAACG-3′ / 5′-TCATTCAGAGCTTTGCCACA-3′); rat Patz1 all variants (5′-CCAGAGCTGTGGGAAAGG-3′ / 5′-TGCACCTGCTTGATATGTCC-3′); rat Cdh1 (5′-CCTGGGACTCCAGTTACAGG-3′ / 5′-CTC.AGACCCTGTGAAAGCTGG-3′); rat Vim (5′-GAATACCGGAGACAGGTGCAG-3′ / 5′-CGGCCAATAGTGTCCTGGTAG-3′).
4
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from cells using TRI-reagent solution (Sigma) following the manufacturer’s protocol. Reverse transcription was performed according to standard procedures using a SensiFAST™ cDNA Synthesis Kit (Aurogene, Rome, Italy). An Applied Biosystems 7900 Real-Time PCR Detection System (ABI 7900HT—Applied Biosystems, Waltham, MA, USA) was used to amplify each cDNA with iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). The PCR conditions were as follows: 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Subsequently, the dissociation curve was calculated to verify the amplification specificity. Each amplification reaction was repeated in duplicate. Primer pairs used were as follows: PATZ1 all variants: 5′-TACATCTGCCAGAGCTGTGG-3′/5′-TGCA CCTGCTTGATATGTCC-3′; ꞵ-ACTIN: 5′-CAAGAGATGGCCACGGCTGCT-3′/5′-TCCTTCTGCATCCTGTCGGCA -3′; CDH1: 5′-GCCTCCTGAAAAGAGAGTGGAAG-3′/5′-TGGCAGTGTCTCTCCAAATCCG-3′; VIM: 5′-GACCAGCTAACCAACGACAAA-3′/5′-GAAGCATCTCCTCCTGCAAT-3′.
The 2−ΔΔCT method was used to calculate the gene expression levels [35 (link)]. Beta-ACTIN was used as the internal normalizer for gene expression.
The 2−ΔΔCT method was used to calculate the gene expression levels [35 (link)]. Beta-ACTIN was used as the internal normalizer for gene expression.
5
Liver RNA Extraction Protocol
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Total RNA was extracted from liver samples using guanidine isothiocynate and phenol (TRI Reagent solution, Sigma-Aldrich, St Louis, MO). Briefly, 50-100 mg of the tissue was homogenized with 1 mL TRI Reagent, and then 0.2 mL of chloroform was added to the homogenate. After centrifugation at 12,000 × g for 15 min, the RNA was precipitated from the aqueous phase by addition 0.5 mL isopropanol followed by centrifugation again at 12,000 × g for 10 min. The pellet was washed with 75 % ethanol and re-suspended in RNase-free water. The RNA then was quantified using a NanoDrop instrument (Thermo Scientific, Wilmington, DE). The integrity of isolated RNA was confirmed using 1 % agarose gel electrophoresis with SYBR Safe™ DNA gel stain (Invitrogen Life Technologies, Grand Island, NY).
6
Quantification of TLR3 and TLR7 mRNA in PBMCs
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Total RNA from PBMCs was extracted using TRI reagent solution (Sigma-Aldrich, St. Louis, MO) as permanufacturer’s instructions. RNA concentration and purity was determined using the BioPhotometer plus (Eppendorf, Hamburg, Germany). Two µg of total RNA was reverse transcribed with Oligo (dT)18 primers using the High Capacity cDNA Archive kit (Applied Biosystems, Carlsbad, CA). Basal expression levels of TLR3 and TLR7 mRNA in PBMC were determined using gene-specific primers and TaqMan probes (FAM-NFQ) (Applied Biosystems, Carlsbad, CA) (Table 1 ). qRT-PCR was performed in triplicate under the following cycle conditions, 2 min at 50°C, 10 min at 95°C and 40 cycles of 95°C for 15 sec and 60°C for 1 min (Applied Biosystems 7500 Real time PCR System, Carlsbad, CA).
7
Quantifying HMGA1 Gene Expression
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Total RNA was isolated using TRI-reagent solution (Sigma) and reverse transcription was performed according to standard procedures (Qiagen, Valencia, CA, USA). qRT-PCR analysis was performed using the following primers:
HMGA1 Fw: 5′-CAACTCCAGGAAGGAAACCA-3′;
HMGA1 Rv: 5′-AGGACTCCTGCGAGATGC-3;
β-actin Fw: 5′- CCAACCGCGAGAAGATGA-3;
β-actin Rv: 5′-CCAGAGGCGTACAGGGATAG -3.
8
Quantitative analysis of gene expression
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Total RNA was extracted from T-75cm2 flasks using Tri Reagent solution (Sigma-Aldrich LLC, St Louis, Missouri). RNA quality (260/280 nm) was determined using a Nanodrop ND 1000 spectrophotometer. One microgram of total RNA was synthesized to 20 µL complementary DNA (cDNA) using the high-capacity cDNA Reverse Transcription Kit from Applied Biosystems (Life Technologies, Saint Aubin, France) according to the manufacturer’s protocol. Quantitative polymerase chain reaction analysis was performed with a QuantStudio 12K Flex Real-Time PCR System (Life Technologies) using TaqMan 6 carboxyfluorescein-labeled probes and a standard thermal cycler protocol (50°C for 2 minutes before the first cycle, 95°C for 15 seconds, and 60°C for 1 minute repeated 45 times). Human TaqMan gene primers for eNOS, endothelin-1, vascular endothelial growth factor-B (VEGF-B), VEGF-receptor2 (VEGF-R2), TGF-β, copper/zinc-superoxide dismutase (Cu/Zn-SOD), manganese-superoxide dismutase (Mn-SOD), interleukin 6 (IL-6), and fibroblast growth factor 2 were from Applied Biosystems. Relative expression levels were calculated with glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein, large, P0 (RPLP0) as internal control genes, and Ct mean was used to analyze the results. Nonirradiated cells were set as 1. Each experiment was performed in triplicate.
9
Gene Expression Analysis by qPCR
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RNA was isolated using TRI reagent solution (Sigma) followed by the on‐column RNeasy mini kit and DNase treatment (Qiagen). cDNA synthesis was performed using the Transcription First Strand cDNA Synthesis Kit (Roche). Quantitative real‐time polymerase chain reaction (qPCR) was performed using the ABI 7900T PCR System (Applied Biosystems). The gene expression was normalized to the expression of housekeeping gene 18S. Relative fold changes were transformed using the comparative threshold cycle (CT) method (the 2‐ΔΔCT method) with the Sequence Detection System V2.4 software (Applied Biosystems). Primer sequences are provided in Supporting Information Table S1 .
10
RNA Extraction and RT-PCR Analysis of GPR15/BOB
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Total RNA was extracted from frozen blocks of synovia using TRIreagent solution (Sigma, Poole, UK) or from isolated monocytes/macrophages using RNAqueous kit (Ambion, Applied Biosystems, Warrington, UK) according to the manufacturer’s instructions. The quantity recovered was determined by spectrophotometry and the integrity was assessed by agarose gel electrophoresis. Total RNA (1 μg) was reverse transcribed using oligo(dT) primers (MWG Biotech, Ebersberg, Germany) and MMLV reverse transcriptase (Promega, Southampton, UK) at 37 °C for 1 h. The reactions were then heated to 70 °C for 5 min to inactivate the enzyme, placed on ice and 60 μl H2O was added. Appropriate dilutions of the resulting cDNA were then used for semi-quantitative PCR using specific primers for GPR15/BOB [F 5′-GTG ATG GAC CCA GAA GAA AC-3′; R 5′-GGA CAG AAG AGT AGG CAA CC-3′ (515 base pairs (bp); GenBank:NM005290); MWG Biotech]. PCR primers were run through a BLAST program to ensure gene specificity. The PCR reactions were normalised against the ribosomal RNA L27 using specific primers [F 5′-GAC GCA AAG CTG TCA TCG TG-3′; R 5′-GCA GTT TCT GGA AGA ACC AC-3′ (344 bp; GenBank:BC007273); MWG Biotech]. The annealing temperature was 57 °C for each primer pair.
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