Veriti pcr system

The wzi locus was sequenced to determine serotypes of the K. pneumoniae strains by searching the Institute Pasteur database (http://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?db=pubmlst_klebsiella_seqdef&page=sequenceQuery).
Virulence genes, wzy-K1, allS, entB, irp2, iroN, iucA, fimH, mrkD, p-rmpA, p-rmpA2, c-rmpA, peg-344, and wzi (Compain et al., 2014 (link); Gu et al., 2018 (link); Russo et al., 2018 (link)), were analysed using a Veriti PCR system (Applied Biosystems). The primers used are shown in Table S1. NTUH-K2044 was used as the positive control. In the subsequent agarose electrophoresis, a proper band in line with the control is regarded as a positive gene.

Total RNA was extracted from the cells using a phenol-free total RNA extraction kit (Norgen Biotek, ON, Canada). Quality and concentration of the extracted RNA was determined by spectrophotometric methods (NanoDrop 8000 UV-Vis Spectrophotometer, Thermo Scientific, Waltham, MA). The levels of expression of the UCHL1 and housekeeping gene (HPRT1) were measured in the glioma cell lines and in the UCHL1 KDs using droplet digital PCR (QX100 ddPCR System, Bio-Rad, Hercules, CA) and Taqman Gene Expression primer/probes sets (Human HPRT1 (FAM-labeled) and UCHL1 (VIC-labeled); Applied Biosystem, Foster City, CA). RT reactions were conducted on a Veriti PCR System (Applied Biosystems) using 100 ng of total RNA, and iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad) as per manufacturer’s guidelines (i.e. 5 min at 25°C, 30 min at 42°C, 5 min at 85°C followed by an end cycle at 4°C). Quantitative PCR reactions were prepared using 2 μL of the RT reactions (diluted 1/10), Taqman Gene Expression Assays, and ddPCR Supermix for probes (Bio-Rad) as per manufacturer’s guidelines. Differences in UCHL1 mRNA expression across samples were determined using QX100 Droplet Reader and QuantaSoft TM Software (Bio-Rad).

Total RNA in lysed lung tissue was extracted using Trizol Reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA) and reversely transcribed to cDNA by applying mouse Moloney leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA). mRNA levels in lung tissue of MAVS, IRF3, NF-κB, IL-6, IL-1β, CYP11B, CYP17A, and CYP21A and of the internal control 18S gene were measured by the Veriti PCR System (Applied Biosystems). The PCR products were fractionated on a 1% agarose gel and visualized by ethidium bromide staining. The band intensity of ethidium bromide fluorescence was measured using an image analysis system (Bio-Rad, Hercules, CA), then quantified with Quantity One analysis software (Bio-Rad, Hercules, CA), and expressed as the ratio to 18S. The sequences of the primers were as follows: 18S, 5′-AGGGGAGAGCGGGTAAGAGA-3′ and 5′-GGACAGGACTAGGCGGAACA-3′; MAVS, 5′-CAGATTGGTCCCAGTAA-3′ and 5′-GCAAGGTCCACAGAGC-3′; IRF3, 5′-AGAGGCTTGTGATGGT-3′ and 5′-GGCTGTTGGAGATGTG-3′; NF-κB, 5′-TTTATCTCGCTTTCGG-3′ and 5′-GCTCCAGTCTGTCCCTC-3′; IL-1β, 5′-GCTGGAGAGTGTGGAT-3′ and 5′-CTTGTGAGGTGCTGATG-3′; IL-6, 5′-CCAACAGACCTGTCTATACCAC-3′ and 5′-GTGACTCCAGCTTATC-3′; CYP11B, 5′-CAGAACTAATGTGTATGT-3′ and 5′-TTGACCAGAGAAGATG-3′; CYP17A, 5′-CTGATACAAGCCAAGAT-3′ and 5′-CTGAAGCCTACATACTG-3′; CYP21A, 5′-CACTTCCTACAGCCTAA-3′ and 5′-CCTCCTCAATGGTTCT-3′.