Isolate 2 rna kit

Quantitative real-time polymerase chain reaction (RT-qPCR) was used to estimate the number of viral RNA genomes. RNA was extracted from cell-associated and cell-free virus using the Isolate II RNA kit (Bioline, Eveleigh, Australia), according to the manufacturer’s instructions. Nucleotide sequences (primers and probe) were directed to a region within the N gene of RSVA [31 (link)]. Primers (forward: 5′-CTC AAT TTC CTC ACT TCT CCA GTG T; reverse: 5′-CTT GAT TCC TCG GTG TAC CTC TGT) were synthesized by Integrated DNA Technologies. The probe (5′-TCC CAT TAT GCC TAG GCC AGC AGC A) was labeled with the 5′ reporter dye 6-carboxy-fluorescein (FAM) and the 3′ quencher dye 6-carboxy-tetramethylrhodamine (TAMRA) by Applied Biosystems. A one-step protocol was used with 10 μL of RNA added to each reaction mixture containing 1× TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems, Scoresby, Australia), 300 nM of each of the primers, and 200 nM of the probe. The amplification profile used was 1 cycle for 5 min at 50 °C and 20 s at 95 °C, followed by 40 cycles for 3 and 30 s at 95 °C and 60 °C respectively. Absolute RNA copies were determined by extrapolation from a standard curve produced using a plasmid carrying the RSVA N gene cDNA [8 (link)].

Primed THP-1 cells were lyzed with TRizol reagent (Invitrogen), 6 h after crystal stimulation, and total RNA was extracted by using the ISOLATE II RNA kit (Bioline, London, UK). First, 500 ng of total RNA were reverse transcripted to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Foster City, California, USA) (LifeECO Thermal Cycler, Bioer Technology, Hangzhou, Binjiang, China). Then, quantitative PCR was performed with 25 ng of cDNA using the SensiFAST SYBR No-ROX Kit (Bioline, London, UK) for 40 cycles (95 °C for 5 s, 60 °C for 30 s) (LightCycler®480 Instrument, Roche Life Science, Penzberg, Germany). Sequences of primers for qPCR are reported in Table 2.

Messenger RNA was extracted from a cell pellet using the Isolate II RNA kit (Bioline, Alexandria) as per the manufacturer’s instructions. In brief, cells were lysed with 350 μL lysis buffer and 3.5 μL β-mercaptoethanol. Lysate was loaded onto an ISOLATE II filter and centrifuged at 11,000 x g in a 2 mL collection tube for 1 min. Then 350 μL of 70% ethanol was added to each collection tube, lysate loaded into an ISOLATE II Mini column with a 2 mL collection tube and centrifuged for 30 s at 11,000 x g. The flow through was discarded and 350 μL Membrane Desalting Buffer was added and centrifuged at 11,000 x g for 1 min. Reconstituted DNase 1 was added to Reaction Buffer at a 1:10 dilution. The solution was mixed and 95 μL was added to each column and incubated at room temperature (RT) for 15 min. The column was washed with 200 μL Wash Buffer 1 and centrifuged for 30 s at 11,000 x g. The Flow through discarded and the column washed twice with Wash buffer 2 with 600 μL for 30 s at 11 000 x g and then 250 μL for 2 min. The column was placed into a 1.5 mL collection tube and eluted with 30 μL RNase-free water by centrifugation at 11,000 x g for 1 min. The RNA yield was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Scoresby) and diluted to a final concentration of 166.6 ng/μL. All samples were frozen at -20°C until required.

For the RNA-Seq study, triplicate wells of 22Rv1 cells were transfected using Lipofectamine 2000 with 30 nM miR-642a-5p or miR-NC, and total RNA extracted from the samples 24 h post-transfection, using the Isolate II RNA kit (Bioline) according to the manufacturer’s instructions. The quantity and integrity of extracted RNA was determined using a 2100 Bioanalyzer (Agilent Technologies), before RNA-Seq analysis using the Illumina HiSeq 2500 at the Australian Genome Research Facility (AGRF; Victoria, Australia). Analysts at AGRF normalized the data with the R Bioconductor ‘EdgeR’ package (www.Bioconductor.org). Briefly, sequence counts were aligned to the genome, background corrected, log₂ transformed, annotated, and a fold change analysis performed to compare treatment groups.
TargetScan (Version 7.2: March 2018) provided metadata on genes downregulated by miR-642a-5p in the RNA-Seq experiment. Gene Set Enrichment Analysis (GSEA) of the RNA-Seq data was performed as previously described^{68 (link)}. The biological pathway targets of genes differentially expressed by miR-642a-5p were determined using Ingenuity Pathway Analysis (IPA, Ingenuity System, Inc. www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis). The RNA-Seq data is available in the Gene Expression Omnibus under Accession Number GSE160736.

Primed THP-1 cells were lysed with TRizol reagent (Invitrogen), 6 h after crystal stimulation, and total RNA was extracted by using the ISOLATE II RNA kit (Bioline). First, 500 ng of total RNA were reverse transcript to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem) (LifeECO Thermal Cycler, Bioer Technology). Then, real time quantitative PCR was performed with 25 ng of cDNA using the SensiFAST SYBR No-ROX Kit (Bioline) for 40 cycles (95°C for 5 s, 60°C for 30 s) (LightCycler®480 Instrument, Roche). Sequences of primers for real time qPCR are listed in Table 1.

RNA was extracted from OS tissue as previously described [43 (link)]. RNA was also extracted from cell lines with the Isolate II RNA kit (Bioline). cDNA was synthesised with the Tetro kit (Bioline) with oligo-(dT) primers. Both SYBR-green and multiplex based probe qPCR were performed on the Stratagene Mx3000P machine (Agilent Technologies) with gene-specific primers (S3 Table). The relative gene expression was normalized to the HPRT housekeeping gene and calculated by the 2^-ΔCT method.

Cells were treated for 24 h with E2 (10 nM), tam (1 µm), or fulv (100 nM); E2-treated cells were hormone-starved for 24 h as described above. Total RNA was isolated using Isolate II RNA Kit (Bioline) and cDNA was then synthesized from 1 µg of RNA using XLA Script cDNA Kit (Quanta BioSciences). SYBR Green PCR Mix (Apex) was used for Real Time qPCR on the ABI Fast 7500 system. Samples were run in triplicates in each experiment and relative mRNA levels were normalized to GAPDH (glyceraldehyde 3-phosphate dehydrogenase). qPCR primer sequences are found in Supplementary Table 9.