RNA was extracted from 5 × 106 cultured cells and DNA removed by on‐column DNase digestion (RNeasy; Qiagen, West Sussex, UK). RNA integrity and concentrations were determined using an Agilent 2100 Bioanalyser (Agilent Technologies, Stockport, UK) before cDNA synthesis from 1 μg RNA using random primers and AffinityScript reverse transcriptase (Agilent). QuantiTect SYBR Green PCR with UNG treatment (Qiagen) was used for amplification and quantification in a Mx3005P QPCR System (Agilent) during 40 PCR cycles. Data were analysed using MxPro QPCR software (Agilent). Changes in mRNA levels were calculated using the 2−ΔΔCt method with GAPDH as the reference gene using primers from Qiagen QuantiTect Primer Assay. Primers for TP63 isoforms (see supplementary material, Table S1 ) were custom synthesised (Sigma‐Aldrich).
Affinityscript reverse transcriptase
Manufactured by Agilent Technologies
Sourced in United Kingdom
AffinityScript reverse transcriptase is a thermostable enzyme used for the conversion of RNA to cDNA. It is capable of efficient reverse transcription of RNA templates at elevated temperatures, allowing for increased specificity and yield of cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Mx3000p light cycler, by Agilent Technologies (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Mxpro qpcr software, by Agilent Technologies (1 mentions)
Quantitect primer assay, by Qiagen (1 mentions)
Mx3005p qpcr system, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (1 mentions)
Sybr green qpcr reagent kit, by Roche (1 mentions)
Mouse monoclonal anti β actin, by Merck Group (1 mentions)
Bioanalyzer rna nano chip, by Agilent Technologies (1 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Hif 1α, by BD (1 mentions)
Hif 2α, by Novus Biologicals (1 mentions)
β actin, by Merck Group (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Direct zol rna microprep kit, by Zymo Research (1 mentions)
T4 rna ligase 1, by Thermo Fisher Scientific (1 mentions)
9 protocols using affinityscript reverse transcriptase
1
Quantitative Real-Time PCR Analysis of TP63 Isoforms
2
RT-qPCR and ELISA quantification of hypoxia markers
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RNA was extracted and quantified by reverse-transcription (RT) real-time quantitative (q) polymerase chain reaction (PCR) as previously described.21 (link),23 (link) In brief, RT was performed with 2 mg total RNA and AffinityScript reverse transcriptase (Agilent), and the cDNA quantified using SYBR Green qPCR reagent kit (Kapa Biosystems, London, UK) in a MX3000P light cycler (Agilent). Transcripts levels were calculated through comparison with calibrated standard curves and normalized to human ribosomal protein L28 mRNA. Primers used for RT-qPCR are listed in Online Supplementary Table S1. Epo protein was detected by ELISA according to the manufacturer's protocol (R&D Systems, Minneapolis, MN, USA). Immunoblotting was performed as previously described21 (link) using the following primary antibodies: mouse monoclonal anti-HIF-1α (#610959; BD Transduction Laboratories, San Jose, CA, USA), rabbit monoclonal anti-HIF- 2α (#PAB12124; Abnova, Taipei, Taiwan), mouse monoclonal anti-HIF-b (D28F3; Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-b-actin (A5441; Sigma Aldrich). Secondary antibodies were HRP-conjugated goat polyclonal anti-rabbit or anti-mouse IgG (#31460 and #31430, respectively; Thermo Fisher Scientific).
3
Quantitative RT-PCR for Gene Expression Analyses
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Quantitative RT-PCR analyses were performed as previously described (26 (link)). Cells were homogenized and RNA purified using the Nucleospin RNA Kit (Macherey-Nagel). RNA was checked for quantity and quality using the Nanodrop 2000 (Thermo-Scientific) and RNA Nano BioAnalyzer chip (Agilent Technologies). Total RNA (1000 ng) was enriched for mRNA using Dynabeads mRNA Purification Kit (Ambion by Life Technologies) prior to cDNA synthesis. For cDNA synthesis, 500 ng of total RNA or mRNA eluted from Dynabeads was reverse transcribed using AffinityScript reverse transcriptase (Agilent Technologies) in a total reaction volume of 23 μl. Reactions were incubated at 25 °C for 10 min, 50 °C for 60 min, and 70 °C for 15 min to terminate the reaction. Gene expression was quantified using the relative standard curve method. Primers and probes were designed using Primer Express Software and synthesized by Eurofins Genomics (Germany). Gene-specific primers and probes are listed in the Supplementary Information .
4
Reverse Transcription and qPCR of miRNAs
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miRNAs were reverse-transcribed into first strand cDNA in a 7.5 μL reaction containing 50 nM of a specific reverse stem-loop primer, 1× RT buffer, 0.25 mM of each dNTP, 3 μL of Affinity Script reverse transcriptase (Agilent Technologies), and 0.25 u/μL RNase out (Invitrogen) as described by Chen et al. [51 (link)]. The reactions were incubated for 30 min at 16 °C, 30 min at 42 °C, 5 min at 85 °C, then 4 °C. The real-time PCR reactions were performed using the Brilliant III Ultra-Fast SYBR QPCR kit (Agilent Technologies). The 10 μL PCR reaction included 1 μL of RT product, 1.5 μM forward primer, 0.7 μM universal primer and 5 μL of 2× SYBR Green PCR mix. The primer sequences are listed in Supplementary Table 1 . The reactions were incubated at 95 °C, 10 min, followed by 40 cycles of 95 °C 15 s and 60 °C 1 min. Specificity of the amplifications was confirmed by the single peak of dissociation curves of the PCR products. All reactions were run in triplicate.
5
Quantifying Immune Cell Markers
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Affinity script reverse transcriptase (Agilent) was used to generate cDNA from 1 RNA μg in 20 µL water, following the manufacturer’s instructions. Quantitative real-time PCR was performed from 2 µL cDNA in BIORAD CFX96 equipment using TaqMan assays for CD14, CD48, MARCKS, GILZ, and PPIB mRNA.
6
Quantitative Analysis of RNA and Protein Expression
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Total cellular RNA was extracted as previously described.39 (link) Total RNA (2 µg) was reverse transcribed using Affinity-Script reverse transcriptase (Agilent, Santa Clara, CA, USA) and complementary DNA (cDNA) levels were estimated by quantitative polymerase chain reaction (PCR) using a SYBR® Green quantitative PCR reagent kit (Sigma-Aldrich) in a MX3000P light cycler (Agilent). Transcript levels were calculated by comparison with a calibrated standard and expressed as ratios relative to ribosomal protein L28 mRNA levels. Immunoblots were performed as previously described.40 (link) Antibodies against the following proteins were used: WISP-2 (Abcam, Cambridge, UK), HIF-1α (BD Transduction Laboratories, Allschwil, Switzerland), HIF-2α (Novus Biologicals, Littleton, CO, USA), Sp1 (Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin (Sigma-Aldrich). Breast cancer tissue microarray analysis has been described previously.8 (link)
7
SARS-CoV-2 Viral RNA Quantification
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Approximately 2x105 cells were lysed in 300 μl Trizol/well and RNA was extracted using the Direct-zol RNA MicroPrep Kit (Zymo Research, R2063). RNA was reverse transcribed into cDNA using Affinity Script reverse transcriptase (Agilent, 600109) according to the manufacturer’s instructions using random primers. Viral RNA was quantified by qPCR using PowerUp SYBR Green master mix (Thermo Fisher Scientific, A25778) or SsoAdvanced Universal SYBR Green Supermix (Biorad Laboratories, 1725272) and specific primers for viral RdRP gene (Fwd: 5ʹ-GTGARATGGTCATGTGTGGCGG, Rev: CARATGTTAAASACACTATTAGCATA-3ʹ), viral N gene (Fwd: 5ʹ-CACATTGGCACCCGCAATC-3ʹ, Rev: 5ʹ-GAGGAACGAGAAGAGGCTTG-3ʹ) or 18S ribosomal RNA (Fwd: 5ʹ-ATGGCCGTTCTTAGTTGGTG-3ʹ, Rev: 5ʹ-GAACGCCACTTGTCCCTCTA-3ʹ). To specifically analyze viral sgmRNAs and gRNA, we designed a common forward primer that binds within the SARS-CoV-2 leader sequence (5ʹ-CCCAGGTAACAAACCAACCAAC-3ʹ) and a reverse primer specific for the ORF1a RNA (5ʹ-CTCGTTGAAACCAGGGACAAG-3ʹ), the mRNA of M (5ʹ-GGTTCCATTGTTCAAGGAGCTT-3ʹ), or the mRNA of N (5ʹ-GTAATGCGGGGTGCATTTCG-3ʹ).
We performed 4 technical qPCR replicates (from the same cDNA) and took the median value for further analysis. To calculate differences in RNA expression we used the ΔΔCT method versus 18S rRNA.
We performed 4 technical qPCR replicates (from the same cDNA) and took the median value for further analysis. To calculate differences in RNA expression we used the ΔΔCT method versus 18S rRNA.
8
HydraPsiSeq: High-throughput RNA sequencing
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To perform HydraPsiSeq, the fragmented RNA (∼800 ng) was dephosphorylated with FastAP thermosensitive alkaline phosphatase (Thermo Scientific), cleaned by Agencourt RNA clean XP beads (Beckman Coulter), and ligated to a 3′ linker using high concentration T4 RNA Ligase 1 (NEB) in a buffer containing dimethyl sulfoxide, ATP, PEG 8000, and RNase inhibitor (NEB). The ligated RNA was cleaned from excess linker using Dynabeads MyOne SILANE beads (Thermo Scientific), and first strand complementary DNA (cDNA) was prepared using the AffinityScript Reverse Transcriptase (Agilent). The RNA was subsequently degraded using 2 μl of 1 M NaOH, and the cDNA was cleaned using Dynabeads MyOne SILANE beads. The cDNA was further ligated to a 3′ adapter using a high concentration T4 RNA Ligase 1 (NEB) and cleaned of excess adapter using Dynabeads MyOne SILANE beads (Thermo Scientific). The adapter-ligated cDNA was PCR enriched using NEBNext high-fidelity (NEB) polymerase (9 PCR cycles), separated on an E-Gel EX agarose gel (Invitrogen), and size selected at the range of 150 to 300 bp (containing ∼30–180 nt corresponding to RNA). The amplicons were gel purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and sequenced in a Nextseq system (Illumina) in paired end mode (20 million reads for each sample).
9
Gene Expression Profiling of Synovial Tissue
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Total RNA was extracted from snap frozen synovia, homogenised in 1 ml of TRI reagent (Sigma, Poole, UK) and purified according to manufacturer instructions. Total RNA (100 ng) was reverse transcribed to complementary DNA using Affinity Script Reverse Transcriptase (Agilent Technologies, Stockport, UK) and random primers, according to the manufacturer's protocol. The reaction was incubated at 25 C for 10 min, then 50 C for 60 min and terminated by incubation at 70 C for 15 min. The cDNA was in a total reaction volume of 28 ml.
Gene expression profiling was performed using custom-made 384 well microfluidic cards (TaqMan ® Array Card, Applied Biosystems, Waltham, MA). Each card consisted of four reference genes (Beta actin [ACTB], Glyceraldehyde 3-phosphate dehydrogenase [GAPDH], Hydroxymethylbilane Synthase [HMBS] and Ubiquitin C [UBC]) and 92 target genes, which were identified as possibly mediating pain through sensitising peripheral nerve terminals (supplementary table 1).
For each tissue sample a reaction mix was made using 100 ml of diluted cDNA (1:4) and 100 ml of TaqMan Universal PCR Master Mix. Reaction mix (100 ml) was loaded into two adjacent ports in the microfluidic card which allowing duplicate runs on a 7900HT Fast Real-Time PCR system (Applied Biosystems). RNA expression values are reported as arbitrary units normalised to reference gene expression.
Gene expression profiling was performed using custom-made 384 well microfluidic cards (TaqMan ® Array Card, Applied Biosystems, Waltham, MA). Each card consisted of four reference genes (Beta actin [ACTB], Glyceraldehyde 3-phosphate dehydrogenase [GAPDH], Hydroxymethylbilane Synthase [HMBS] and Ubiquitin C [UBC]) and 92 target genes, which were identified as possibly mediating pain through sensitising peripheral nerve terminals (supplementary table 1).
For each tissue sample a reaction mix was made using 100 ml of diluted cDNA (1:4) and 100 ml of TaqMan Universal PCR Master Mix. Reaction mix (100 ml) was loaded into two adjacent ports in the microfluidic card which allowing duplicate runs on a 7900HT Fast Real-Time PCR system (Applied Biosystems). RNA expression values are reported as arbitrary units normalised to reference gene expression.
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