Cfx manager tm software version 3

Total RNA was extracted from mature, fully differentiated 3T3-L1 adipocytes on day 8 using the TRIzol (Invitrogen, Carlsbad, CA, USA) method [82 (link)]. DNA contamination was removed using the DNase treatment (Promega, Madison, WI, USA). Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA) was used to determine the concentration and purity (260/280) of extracted RNA, and RNA integrity was confirmed using 1.2% agarose gel. The gene expression analysis was performed using the Bio-Rad CFX96^TM Real-Time System. Primers used in the real-time quantitative polymerase chain reaction (qPCR) were designed using NCBI primer blast, and purchased from IDT Technologies (Coralville, IA, USA). The efficiency of all primers was within the acceptable range of 90–110%. Primer sequences are presented in Supplementary Table S1. Amplification was carried out using iQ SYBR Green Supermix (# 1708880, Bio-Rad, Hercules, CA, USA), and a reaction volume was 10 μL with 50 ng of cDNA per reaction as per our previous publications [83 (link)]. Data analyses were carried out using the CFX Manager TM Software, version 3.0 (Bio-Rad, Hercules, CA, USA). The delta Ct values for each gene of interest were obtained, and the mRNA expression of target genes was normalized to RPLP0 as the reference gene, a large ribosomal protein. The expression of target genes was calculated using the ΔΔCt method [84 (link)].

Total RNA was extracted^{44 (link),45 (link)} and used for quantitative real-time PCR which was conducted using a C1000 thermal cycler/Bio-Rad CFX96 Touch real-time PCR detection system (Bio-Rad, Hercules, California, USA) and Optical System Software version 3.0a (Bio-Rad Laboratories). For transcript measurements, the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd) was used. The amplification condition was: one cycle at 95 °C for 1 min, followed by 40 cycles of 95 °C (for 15 s), 58 °C (for 15 s), and 72 °C (for 15 s), and plate reading after each cycle. The name and the sequence of the gene-specific primers for Arabidopsis OAF, internal OAF (inOAF), uOAF, CAD9, miR847A, and for orchids (PaOAF of Phalaenopsis, CsOAF of Cymbidium and CaOAF of Cattleya) were listed in Supplementary Table 3. The data were analyzed using CFX Manager^TM Software (Version 3.0; Bio-Rad Laboratories, Inc.). The transcript levels for genes were determined using three replicates and were normalized using reference housekeeping genes UBQ10 for Arabidopsis (AtUBQ10)^{46 (link),47 (link)} and ACTIN for orchids (PaACT4 for Phalaenopsis, OnACT for Cymbidium and Cattleya)^{44 (link),47 (link)} (Supplementary Table 3).

Molecular identification of B. anthracis was performed using qualitative real-time PCR. The method is based on the amplification of specific DNA sequences using three pairs of specific primers [11 (link)] as follows: R1/R2 primers, specific for the BA813 gene located on the B. anthracis chromosome; PAG 23/24 primers, specific for the protective antigen (PA) gene located on the virulence plasmid pXO1; and CAP 57/58 primers, specific for the capsule (CAP) gene located on the virulence plasmid pXO2. Each 20 μl reaction mixture contained 1x Sso Advanced TM SYBR® Green Supermix (BIORAD), 300 nM each forward and reverse primer, and approximately 10 ng DNA template. The amplification was performed using the CFX Connect Real Time PCR Detection System (BIORAD). A melting curve was generated at 0.5°C increments between 65°C and 95°C, and was analyzed by CFX Manager TM Software, Version 3.0 (BIORAD).