Qpcr master mix plus low rox

Total RNA was extracted by RNeasy Mini Kit (Qiagen, Hilden, Germany). according to the manufacturer`s protocol. One microgram of total RNA was transcribed into cDNA by M-MLV Reverse Transcriptase (Invitrogen). Afterward, Taqman^® or SybrGreen^® analyses were performed using the qPCR Master Mix Plus Low ROX (Eurogentec, Seraing, Belgium) or 2X Takyon for SYBR Assay-ROX, respectively, and the Applied Biosystems 7500 Real Time PCR System. The following primer and probe pairs were used: NAMPT (forward: 5′-GCA-GAA-GCC-GAG-TTC-AAC-ATC-3′; reverse: 5′-TGC-TTG-TGT-TGG-GTG-GAT-ATT-G-3′; probe: 5′-TGG-CCA-CCG-ACT-CCT-ACA-AGG-TTA-CTC-AC-3′), β-actin (forward: 5′-CGA-GCG-CGG-CTA-CAG-CTT-3′; reverse: 5′-CCT-TAA-TGT-CAC-GCA-CGA-TTT-3′; probe: 5′-ACC-ACC-ACG-GCC-GAG-CGG-3′), TATA-box-binding protein (TBP; forward: 5′-TTG TAA ACT TGA CCT AAA GAC CAT TGC-3′: reverse: 5′-TTC GTG GCT CTC TTA TCC TCA TG-3′; probe: AAC GCC GAA TAT AAT CCC AAG CGG TTT G-3′) and hypoxanthine phosphoribosyltransferase (HPRT; forward: 5′- GGC AGT ATA ATC CAA AGA TGG TCA A-3′; reverse: 5′-GTC TGG CTT ATA TCC AAC ACT TCG T-3′; probe: 5′- CAA GCT TGC TGG TGA AAA GGA CCC C-3′).
After reverse transcription with miScript II RT, micro-RNA-34a was quantified using miScript primer assays for miR34a-1 and Hs_RNU6_2_1 for normalization with miScript SYBR Green PCR kit (Qiagen, Hilden, Germany).

For total RNA extraction 0.3 × 10⁶ HepG2 cells per well were seeded in 6 well plates. Total RNA was extracted by RNeasy Mini Kit (Qiagen) according to manufacturer’s protocol. 1000 ng of total RNA was transcribed into cDNA by M-MLV Reverse Transcriptase (Invitrogen). Afterwards, Taqman® analyses were performed using the qPCR Master Mix Plus Low ROX (Eurogentec) and the Applied Biosystems 7500 Real Time PCR System. NAMPT (forward: 5′-GCA-GAA-GCC-GAG-TTC-AAC-ATC-3′; reverse: 5′- TGC-TTG-TGT-TGG-GTG-GAT-ATT-G-3′; probe: 5′-TGG-CCA-CCG-ACT-CCT-ACA-AGG-TTA-CTC-AC-3′) was normalised to the mean of the housekeeping gene ß-actin (forward: 5′-CGA-GCG-CGG-CTA-CAG-CTT-3′; reverse: 5′-CCT-TAA-TGT-CAC-GCA-CGA-TTT-3′; probe: 5′-ACC-ACC-ACG-GCC-GAG-CGG-3′).

E. faecium Case1VVE-S and the in vitro-generated vancomycin-resistant mutant, as well as control BM4147, were grown in 15 ml of BHI while recording medium turbidity with a spectrophotometer. Total RNA was extracted from 2 ml of mid-log-phase cultures by using an RNeasy Protect bacterial minikit (Qiagen) according to the manufacturer's instructions with 20,000 U of mutanolysin (Sigma-Aldrich) added to the lysis step. Contaminating DNA was removed by using the Heat&Run gDNA removal kit (Arcticzymes, Tromsø, Norway) and cDNA produced from 100 ng of RNA by using the high-capacity RNA-to-cDNA kit (Applied Biosystems) according to the manufacturer's instructions. Primers and TaqMan probes for real-time PCRs are listed in Table S2 in the supplemental material, and PCR was performed using qPCR Mastermix Plus Low ROX (Eurogentec, Liege, Belgium) according to standard protocols supplied by the manufacturer. Reactions without reverse transcriptase were used as a control for DNA contamination after DNase treatment. All qPCRs were performed in triplicates. ΔRn threshold was standardized for all reactions. The Livak method was used to calculate the fold changes (41 (link)).

For qRT-PCR gene expression analysis, total RNA was extracted using the RNeasy Mini Kit or RNeasy Micro Kit (Qiagen) with on-column DNase digest. Reverse transcription was performed using the First-Strand cDNA Synthesis Kit (GE Healthcare). Quantitative RT–PCR was performed using qPCR MasterMix Plus Low ROX (Eurogentec) and TaqMan Gene Expression Assays (Thermo Fisher Scientific). The samples were run on the ViiA 7 thermocycler (Thermo Fisher Scientific) using standard cycling parameters provided by the manufacturer. The relative expression of genes of interest was calculated by the ∆Ct and ∆∆Ct method with GUSB used as a reference gene. For gene expression analysis after the differentiation towards hepatic progenitors, UBC was used as a second reference gene. References for the TaqMan assays can be found in Supplementary Table S3.