Lifeeco thermal cycler

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.

The 16S rRNA gene was amplified from genomic DNA by PCR using the primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’), 1492R (5’-TACGGYTACCTTGTTACGACTT-3’), 337F (5’-GACTCCTACGGGAGGCWGCAG-3’), and 1391R (5’-GACGGGCGGTGTGTRCA-3’). PCR was performed with 25 ng of genomic DNA template using 2X KAPA HiFi HotStart ReadyMix (Roche) and 0.3 μM each primer in a LifeECO thermal cycler (Bioer Technology, Hangzhou, China), with the following thermocycling programme: denaturation at 95°C for 3 min; 30 cycles of 98°C for 20 s, 60°C for 15 s, and 72°C for 30 s; and a final elongation at 72°C for 1 min. The 16S rRNA gene sequences were determined with a 3130xI Genetic Analyzer (Applied Biosystems, Foster, CA, USA) using amplified fragments and PCR primers. Partial rRNA sequences of approximately 1.5 kb were compared to the reference sequences of the Mycobacterium genus in the SILVA database, using blastn. Sequences showing ≥98.7% identity were selected.

Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and first-strand cDNA was synthesized using the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) according to the manufacturer’s instructions. The polymerase chain reaction (PCR) was performed using the LifeECO Thermal Cycler (Bioer Technology, Zhejiang, China), and qRT-PCR was performed using Faster Essential DNA Green (Roche Diagnostics, Mannheim, Germany) and the Light Cycler® Nano (Roche). The primers used for RT-PCR and qRT-PCR are listed in the Supplementary table and are mainly quoted from the study performed of Takahashi et al.^{1 (link)}.

Biopsies were stored in −80 °C prior to RNA extraction. For samples embedded in Tissue-Tek, two scalpels were used to cut the tissue out as it was thawing. The remaining samples had been frozen directly in the vial. Homogenization was undertaken using the Precellys 2 mL soft tissue homogenizing ceramic beads kit (CK14, Bertin Instruments, Montigny-le-Bretonneux, France) and Precellys 24 tissue homogenizer. The tissue was cut into quarters using a scalpel and then placed in the Precellys tubes, adding 600 µL kit buffer RLT Plus followed by placing tubes on ice. Aliquots (600 µL) of homogenized sample were used with the Qiagen Allprep DNA/RNA/miRNA kit (Qiagen, Hilden, Germany) and extraction was performed according to supplier instructions. The extracted RNA was eluted once and quality control was performed using NanoDrop Lite (Thermo Fisher Scientific, Waltham, MA, USA) whereupon extracted samples were placed in −20 °C. The GoScript reverse transcription mix, Oligo(dT) (Promega, Madison, WI, USA), was used for the conversion of RNA from the biopsy samples, diluted to contain 15 ng/µL total RNA, into cDNA and the reverse transcription was performed in the LifeECO thermal cycler (BIOER, Hangzhou, China). The following conditions were used: 10 min at 25 °C, 120 min at 37 °C, followed by termination at 85 °C for 5 min and then cooling of the samples.

PCR amplification of DNA was performed using a LifeECO thermal cycler (version 1.04; Bioer Technology) and Quick Taq HS DyeMix DNA polymerase (Toyobo). Sanger sequencing was entrusted to Fasmac. PCR products were purified using a high pure PCR product purification kit (Roche). Customized oligonucleotide primers (see Table S1 in the supplemental material) were purchased from Fasmac and Hokkaido System Science. Chromosomal and plasmid DNA molecules were extracted with a DNeasy blood and tissue kit (Qiagen) and a high pure plasmid isolation kit (Roche), respectively.

Paternity testing was performed using the Microreader^™ 21 ID System (Microread Genetics, Beijing, China) and Microreader^™ 29Y ID System (Microread Genetics, Beijing, China). The genomic DNA extracted from all subjects was amplified using Life ECO Thermal Cycler (Bioer Technology, Hangzhou, China) according to the manufacturer’s instructions. Autosomal STR genotyping was performed using the 3130 Genetic Analyzer system (Thermo Fisher Scientific). The genotyping results were analysed using GeneMapperTM v3.0 software (Thermo Fisher Scientific).