Rotor gene q 5plex platform

Total RNA samples were isolated from HT-29 cells (control, transfected or transduced), using the MagNA Pure Compact Instrument (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instructions (the procedure included DNase treatment). Purified RNA samples were quantified with Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and sample quality was estimated by the calculation of RNA Integrity Number (RIN), using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA samples with a RIN higher than 8.0 were used for subsequent analysis. Poly (A⁺) mRNA fraction was isolated from 1 μg of total RNA samples using NEBNext poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA). The cDNA library preparation was carried out using NEBNext Ultra Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina (New England Biolabs, Ipswich, MA, USA) according to manufacturer’s instructions. The quality and concentration of cDNA libraries were assessed as described above; cluster densities were optimized by qPCR, using Rotor-Gene Q 5 plex platform (Qiagen, Limburg, The Netherlands). Obtained cDNA libraries were sequenced in triplicate on the Illumina NextSeq 500 platform under the 2 × 43 bp paired-end model, yielding 170 M mapped reads per experiment.

A reaction containing 10 μL of QuantiFast SYBR Green PCR (Qiagen, USA), 0.5 mM of each primer, and 2 μL of sample was used for M. tuberculosis DNA detection directly from the CSF sample. Thermocycling was performed in the PCR system in the real time Rotor-Gene Q 5plex Platform (Qiagen, USA) using the following conditions: an initial cycle of five minutes at 95°C, followed by 45 cycles of 30 seconds at 94°C and 45 seconds at the respective annealing temperature for IS6110 (65°C), hsp65 KDa (62°C), and MPB64 (61°C). At the conclusion of cycling a melting step ranging from 72°C to 95°C with an increase of 0.5°C per second was added for each gene.

RNA was isolated via an RNeasy Mini Kit (Qiagen, Hilden, Germany) and transcribed to cDNA via the SuperScript IV First-Strand Synthesis kit (Thermo Fisher Scientific, Rockford, IL, USA). Both kits were used in accordance with the manufacturer’s instructions. qPCRs were performed in quadruplicate using the QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany) on the Rotor-Gene Q 5plex platform (Qiagen, Hilden, Germany).
Due to their stable expression levels in a variety of tissues, the housekeepers GAPDH and β-actin were used as the internal control.
The following primers were used in the experiments: β-actin forward: 5′ CCA CCA TGT ACC CTG GCA TT 3′ and reverse: 5′ TCA GGA GGA GCA ATG ATC TTG A 3′; GAPDH forward: 5′ CCT GCA CCA CCA ACT GCT TA 3′ and reverse: 5′ TGG CAT GGA CTG TGG TCA TG 3′; and brachyury forward: 5′ GTG ACC AAG AAC GGC AGG AG 3′ and reverse: 5′ TAC TTC CAG CGG TGG TTG TC 3′.
The expression levels were calculated based on the 2^−ΔΔCT method.

All patients were diagnosed as COVID-19-positive by PCR test for SARS-CoV-2 performed on nasopharyngeal swab and/or on autoptic samples. Postmortem swabs from different organs were pre-treated with 1:1 ATL lysis buffer, and total nucleic acids (DNA/RNA) were extracted using the QIAsymphony^® instrument with QIAsymphony^® DSP Virus/Pathogen Midi Kit (Complex 400 protocol) according to the manufacturer’s instructions (QIAGEN, Qiagen, Hilden, Germany). SARS-CoV-2 qualitative reverse-transcriptase–polymerase chain reaction (RT-PCR) testing using RealStar^® SARS-CoV-2 RT-PCR Kit 1.0 targeting E and S genes (Altona Diagnostic GmbH, Hamburg, Germany) was performed according to the manufacturer’s instructions, on the QIAGEN Rotor-Gene Q 5 PLEX platform. The number of cycles (Ct values) needed to detect the viral RNA was considered to better understand the relative amounts of genetic material present in the samples (the lower the Ct value, the more viral RNA was in the sample). The threshold of Ct value under which a sample was interpreted as positive was 40.

Bisulfite-modified plasma DNA was amplified by a MethyLight assay [42 (link),43 (link)] using a Rotor-Gene Q 5plex Platform (Qiagen, Hilden, Germany). RPRML locus-specific amplification (+11 to +152 from the TSS) was performed using the following primers and fluorescent reporter probe: forward, 5′-TTCGGTTTTAGTTTTTGCGTC-3′; reverse, 5′-AACCGACTCCTACGATACGAA-3′; probe, 5′-FAM-CGGTTCGAGAGCGCGTAGGTAGTTA-TAMRA-3′. The MethyLight reaction was performed using 4 µL bisulfite-modified plasma DNA, 1× LightCycler FastStart DNA Master HybProbe (Roche, Basel, Germany), 0.6 µM each primer, and 0.2 µM oligonucleotide probe. The thermal profile was: 95 °C for 10 min, followed by 45 cycles at 95 °C for 5 s and 60 °C for 55 s. Amplification of a methylation-independent sequence from the MYOD1 gene was used as a control of DNA input, as described elsewhere [44 (link)]. For absolute quantification, a standard curve was prepared by serial dilutions of a synthetic double-stranded RPRML DNA fragment (Integrated DNA Technologies, Coralville, IA, USA) starting at 1 ng/µL. A reference dilution was included in each plate for normalization between plates. Threshold cycle (Ct) values obtained from plasma samples were subsequently interpolated on the standard curve to determine the number of DNA copies/mL plasma using Rotor-Gene Q Series Software 2.3.5 (Qiagen, Hilden, Germany, RRID: SCR_015740).

M. tuberculosis quantification by TB-MBLA was performed as described by Gillespie et al. (30 (link)). In summary, M. tuberculosis RNA in 1 ml of homogenized sputum preserved in 4 ml of guanidine thiocyanate (GTC) at −80°C was extracted using the RNA pro kit (FastRNA Pro BlueKit; MP Biomedical, CA, USA) as instructed by the manufacturer. The extract was treated with DNase I enzyme (TURBO DNA-free kit; Ambion, CA, USA) to remove DNA from the dead cells. The M. tuberculosis 16S rRNA, a biomarker for viable cells, was amplified and quantified by RT-qPCR using specific primers and probes. The Cq was translated to bacterial load (estimated CFU per ml [eCFU/ml]) using a standard curve on a Rotor gene Q 5plex platform (Qiagen). The cutoff for TB-MBLA positivity was a 30 Cq value that corresponds to 1.0 log₁₀ eCFU/ml, beyond which the test was considered negative (16 (link), 30 (link)).

Total RNA was purified from C2C12 cells and GA muscles using Qiagen RNeasy mini kit and Qiagen RNeasy Fibrous Tissue mini kit, respectively. The isolated RNA (2 µg) was reverse-transcribed using a PrimeScript cDNA synthesis kit (Takara, Shiga, Japan). qPCR was performed using the Rotor-Gene Q 5plex Platform (Qiagen) with TB Green Premix Ex Taq II (Takara). Primer sequences were described in Supplement (Table S1).