Rotor gene thermal cycler

DNA samples were amplified using LAMP and detected using SYTO9 in real time on a Qiagen (Hilden, Germany) RotorGene thermal cycler acquiring to the green channel unless otherwise specified. All reagents were supplied by Sigma Aldrich (Poole, UK) unless otherwise stated. The reaction chemistry for LAMP and amplification detection was 1X isothermal buffer (New England Biolabs Inc, Massachusetts, United States), 300 micromolar each deoxynucleotide triphosphate (dNTP), 0.8 micromolar Betaine, 0.32 units per microlitre Bst polymerase v2.0 warm start (NEB), 0.5 micromolar SYTO9 Green, 0.8 micromolar each LAMP primer, 0.4 micromolar each Loop primer, 0.2 micromolar each displacement primer and molecular grade water for a reaction volume of 20 microlitres. The parameters were set for 60 cycles of 60 seconds at 60 degrees C unless otherwise stated. Temperature melt analysis provided data between 60 and 95 degrees C. Results were analysed on RotorGene 6000 software v1.7 and Microsoft Excel. The threshold was set at the mid point between the background fluorescence and quenched fluorescence from the positive samples. The default threshold value was −0.05 on the normalised fluorescence scale. Standard normalisation was used in the RotorGene software to take the average background from the first five cycles to provide a value by which sample data points are divided.

Total RNA was extracted from Aag2 cells and treated with DNase I using TURBO DNase (ThermoFisher Scientific). cDNA (2μg) was synthetised using SuperScript™ III Reverse Transcriptase (ThermoFisher Scientific) followed by qPCR using QuantiFast SYBR Green PCR mix (Qiagen) in accordance with the manufacturer’s instructions. RT-qPCRs were carried out using a Rotor-Gene thermal cycler (QIAGEN). For qPCR, cDNA was diluted in 1:5 with UltraPure DNase/RNase-free water (Invitrogen). Two microlitres of the diluted cDNA was used in 10 μL qPCR reactions with both forward and reverse gene-specific primers (10 μM) with an initial 95 °C 5 min, and 40 cycles of 95 °C 20 s, 65 °C 15 s, 72 °C 10 s. Each reaction was run with at least three biological replicates, each with three technical replicates. The RPS17 gene was used for normalizing data. Primer sequences are provided in Supplementary Table 2.
For MeRIP-RT-qPCR, EpiMark N⁶-Methyladenosine Enrichment Kit was first used to conduct MeRIP on total RNA harvested from DENV-infected Aag2 cells according to the manufacturer’s instructions (New England BioLabs). This was followed by RT-qPCR as above. Positive and negative controls for m⁶A were provided in the kit.

Bacterial DNA from stool samples was extracted using RIDA^® Xtract kit following the manufacturer’s instructions (cat. no.:PGZ001; R-Biopharm AG, Darmstadt, Germany). For bacteria quantity estimation, qPCR was performed with the use of RIDA^®GENE Akkermansia muciniphila, RIDA^®GENE Faecalibacterium prausnitzii, and RIDA^®GENE Clostridium difficile kits according to the manufacturer’s protocol (cat. nos.: PG0145, PG0155, and PG0835, respectively; R-Biopharm AG, Darmstadt, Germany). The total reaction volume was 25 μL containing 19.9 μL reaction mix, 0.1 μL Taq Polymerase, and 5 μL DNA extract. Samples were treated as follows: initial denaturation (1 min, 95° C), 45 cycles of denaturation (15 s, 95 °C), and annealing/extension (30 s, 60 °C). The standard curve was generated with DNA standard A (5 × 10² copies/reaction), DNA standard B (5 × 10⁴ copies/reaction), and DNA standard C (5 × 10⁶ copies/reaction). The fluorescence measurements were performed in the RotorGene thermal cycler (QIAGEN, Manheim, Germany). The final number of bacteria/gram of stool was obtained by multiplying by 200 due to the dilution factor of the stool sample during extraction. The reference values are presented in Table 2.

qPCR was performed in triplicate for at least 2 independent experiments with Quantitect primers (Qiagen, Valencia, CA, USA) on a Rotor-Gene thermal cycler (Qiagen) or an iCycler (Bio-Rad, Hercules, CA, USA). Data were normalized to Hprt expression and are represented as relative mean expression ± sd of independent experiments, the number of data points indicating the number of mice or samples analyzed.

Fluorescently labelled quenched primers^{13 (link)} were synthesised by Sigma with the fluorophore attached to the 5’ end of the oligonucleotide without further adaptation. JOE (6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein) selected for detection on the yellow channel of a Qiagen (Hilden, Germany) RotorGene thermal cycler. Labelled FIP LAMP primers for 35Sp and NOSt purified to the HPLC level (Table 3).Table 3

LAMP quenched fluorescent primers Modified 35S promoter and NOS terminator FIP primers with JOE (6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein) attached to the 5’ end^{13 (link)}.

Target, Type, Notation	Lgth	Primer Sequence (5’ to 3’)
35Sp, LAMP, JOE-FIP	45	[JOE]CCACGTCTTCAAAGCAAGTGG-TTTT-GGATAGTGGGATTGTGCGTC
NOSt, LAMP, JOE-FIP	46	[JOE]GCATGACGTTATTTATGAGA-TTTT-TCGCGCTATATTTTGTTTTCTA

The quenched fluorescent LAMP primer was synthesised and HPLC purified by Sigma Aldrich (Poole, UK).

Real-time PCR (Rotor-Gene thermal cycler, Qiagen) was used to determine the gene expression profiles of sod3, psph, g6pc3, hba3, alas2, fah, psat1, and pygm using RNA extracted from the brain. The primers for these genes are presented in Supplementary Table S2. PCR was run for 35 cycles as follows: incubation at 95°C for 5 min, annealing at 95°C for 5 s, and extension at 60°C for 10 s. Expression levels were calculated based on the ^ΔΔCT method. Details are provided in the Supplementary Methods.