Cfx96 real time pcr thermal cycler

Nucleic acids were extracted from all samples using Microlab Nimbus IVD (Seegene Inc.), and RNAs were used for cDNA synthesis using cDNA Synthesis Premix (Seegene Inc.). The samples were tested by using Anyplex II RV16 detection kit (Seegene Inc.) according to the manufacturer's instructions. The assay was used to detect Flu-A, Flu-B, RSV A, RSVB, AdV, HMPV, HCoV-229E, HCoV-NL63, HCoV-OC43, PIV-1, PIV-2, PIV-3, PIV-4, HRV, HEV, and HBoV. Reaction mixtures for virus detection were divided into two panels: A and B. Each panel was used to detect 8 viruses with appropriate controls. Two types of DNA and 14 types of RNA viruses were amplified and detected by using CFX 96 Real-Time PCR Thermal cycler (Bio-Rad). Seegene Viewer software was used to analyze the amplification results. The study was approved by the Research and Ethical Committee of King Abdul-Aziz Medical City, Riyadh.

We prepared RNA from sorted bone marrow and liver samples using the RNeasy Kit (Qiagen) according to the manufacturer’s instructions. We synthesized cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher). Primers are listed in Supplementary file 1a. qPCR was conducted by iQ SYBRGreen Supermix using the BioRad CFX96 Real-Time PCR Thermal Cycler. Target gene mRNA concentration was normalized to Gapdh.

Saturated E. coli K-12 cultures from a single colony were diluted to 0.01 OD₆₀₀ in LB media and grown at 37°C to an OD₆₀₀ of 0.5, after which MMS (5 mM) or mechlorethamine (200 μM) was added and cultures grown at 37°C for an additional 2 hr. Cultures were lysed with TRIzol reagent and the RNA extracted with phenol-chloroform and precipitated with isopropanol/ethanol as in (28 ). Residual genomic DNA was removed from the RNA by treatment with DNase I (New England BioLabs). The RNA was re-extracted from the reaction mixture and quantified by absorbance at 260 and 280 nm. The specificity of the primers and quality of the RNA was verified by agarose gel analysis of RT-PCR products (Supplementary Figure S6F). cDNA synthesis and qPCR were performed in a single step reaction using the iTaq Universal SYBR Green One-Step Kit (Bio-Rad) on a BioRad CFX-96 real-time PCR thermal cycler. The housekeeping gene used was gapA (Eco GAPDH). The results from the qPCR experiments were performed on three biologically replicated RNA extractions from both MMS and mechlorethamine treatments. The fold expression change was calculated using the formula: (fold expression change) = 2^−ΔΔCt, where Ct is the cycle threshold for amplification above baseline, ΔCt = Ct (gene of interest) – Ct (housekeeping gene), and ΔΔCt = ΔCt (treated sample) – ΔCt (untreated sample).

Primary hepatocytes were suspended in 15 mL tubes at 1.2 × 10⁶ cells per 10 mL of attachment medium or seeded in 100 mm petri dishes at 1.2 × 10⁶ cells per dish. RNA was isolated from cells 0, 2, 8, 24, and 48 h post‐isolation using Qiagen RNeasy kits (Toronto, Ontario), with three biological replicates, according to manufacturer's instructions. RNA quality was assessed by the Agilent RNA ScreenTape assay (Mississauga, Ontario) using the Agilent 2200 Tapestation System (Mississauga, Ontario) and all samples achieved RIN^e quality scores of at least 8.5. cDNA was synthesized using Qiagen RT² First Strand kit (Toronto, Ontario) according to manufacturer's instructions. cDNA was prepared and applied to Qiagen Mouse Drug Metabolism RT² profiler PCR arrays (catalog #PAMM‐002Z) (Toronto, Ontario) (Supporting Information Table SVI). The C_t values were determined using a BioRad CFX96 real‐time PCR thermal cycler (Mississauga, Ontario). A C_t cut‐off of 35 was applied.

Vaginal lavage fluid was collected as described in reference 38 (link) and filtered through 0.22-μm Spin-X centrifuge tube filters (Costar) to remove contaminants. Triplicate log-phase cultures of USA300 were pelleted and resuspended in filtered lavage fluid. Following a 2-h incubation at 37°C, bacteria were collected by centrifugation, resuspended in TRIzol, and lysed by bead beating, and RNA was isolated using the Direct-zol RNA MiniPrep Plus kit, as described above. RNA was treated with Turbo DNase (Invitrogen) to remove contaminating DNA. cDNA was generated using the Quanta cDNA synthesis kit (Quanta Biosciences), and qPCR was performed using PerfeCTa SYBR green reagent (Quanta) and a CFX96 real-time PCR thermal cycler (Bio-Rad). Fold changes were calculated using the Livak method (116 (link)).

The stability of expression of the candidate housekeeping genes (Table 1) was analyzed by Genorm (Vandesompele et al., 2001 (link)), using the RefFinder web application (http://www.ciidirsinaloa.com.mx/RefFinder-master/?type=reference) (Fig. 1). The expression of target genes was normalized to the most stable gene (β-actin). Reactions were performed in a CFX96 real-time PCR thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) using 96-well plates.
A qPCR master mix was prepared (2X concentration = 7.5 ul/reaction) in one batch for all PCR reactions and store in aliquots at −20 °C. Each 2X reaction comprised 1.5 µL 10X Reaction Buffer, 0.75 µL 50 mM MgCl₂, 0.3 µL 10 mM dNTPs, 0.75 µL EvaGreen® 20X (Biotium, Hayward, CA, USA), 0.1 µL Invitrogen® DNA Polymerase Recombinant (Life Technologies, Carlsbad, CA, USA) and 5.9 µL ultrapure water to 9.3 µL.
Before PCR, each aliquot of the 2X master mix was thawed, and 0.35 µl of each 10 µM primer (forward/reverse) was added to reach 10 µl. Then 10 µl of mix were placed in each plate well and 5 µl of the corresponding cDNA were added (15 µl per reaction).