Total RNA was prepared from fresh PDAC tissues and cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. qRT-PCR was conducted in a 96-well real time PCR system (Bio-Rad Inc., Hercules, CA) using a SYBR® Premix Ex Taq kit (Takara Bio Inc., Shiga, Japan). GAPDH was used to normalize gene expression. The primers are shown in Supplementary Table s2 . Relative gene expression was calculated from the qRT-PCR data using the 2 −ΔΔCT method.
96 well real time pcr system
Manufactured by Bio-Rad
Sourced in Japan
The 96-well real-time PCR system is a laboratory instrument designed for performing quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is capable of processing 96 samples simultaneously, allowing for high-throughput nucleic acid quantification.
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5 protocols using 96 well real time pcr system
1
Total RNA Extraction and qRT-PCR Analysis
2
Chromatin Immunoprecipitation and qPCR Analysis
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ChIP-qPCR was carried out in the Bio-Rad 96 well Real-Time PCR System and 1 μL of each ChIP-DNA and Input-DNA dilution from ChIP assay with the Hieff® qPCR SYBR Green Master Mix (YEASEN, Lot:H7901050) and 200 nM primers following the manufacturer’s instructions. Dissociation curve analysis was performed for verification of product homogeneity. The gene-specific primer pairs used for ChIP-qPCR for czcR, phzA1 and oprD are RT-czcR-F/RT-czcR-R, RT-phzA1-F/RT-phzA1-R, RT-oprD-F/RT-oprD-R, respectively (Table S2 ). The coding sequence of phoB (RT-phoB-F/RT-phoB-R) was used as a negative control. ChIP-DNA enrichment levels of interest genes were calculated by the relative quantification method (2−ΔΔCt method) [36 (link),37 (link),38 (link),39 (link)] and reported as fold-change. The statistics comparison was done by Student’s two-tailed t-test in GraphPad Prism (version 7.0).
3
Quantification of Gene Expression in Arbuscular Mycorrhizal Fungi
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Total RNA was extracted from different tissues of R. irregularis and mycorrhizal roots using Fungal RNA Kit (OMEGA‐R6840) according to the manufacturers' instructions, and the first‐strand cDNA synthesis was initiated with the HiScript III reverse transcriptase (Vazyme‐R323‐01, Nanjing, China). Real‐time qRT‐PCR reactions were performed in a 96‐well Real‐time PCR system (Bio‐Rad). All the reactions were performed with three technical replicates of three biological replicates. The R. irregularis RiEF1a, A. sinicus AsActin, N. benthamiana NbtEF1a were used as the endogenous reference gene for normalization, respectively. Relative expression levels of target genes were computed by the 2−ΔΔCt method of relative quantification. A list of gene‐specific primers used for real‐time qRT‐PCR is given in Table S2 .
4
Quantitative Analysis of RNA Expression
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All tissues were confirmed by pathological analysis and immediately frozen in liquid nitrogen and stored at -80° C. According to the manufacturer's instructions, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from TTs, NATs, and PAC cell lines. qRT-PCR was performed using a SYBR® Premix Ex Taq kit (Takara Bio Inc., Shiga, Japan) and a 96-well real-time PCR system (Bio-Rad Inc., Hercules, CA, USA). GAPDH or U6 snRNA was used as a loading control. The primers for miR-1252-5p were purchased from Sangon Inc., (Shanghai, China), and the details of the primers for GAPDH, NEDD9, E-cad, N-cad, ZEB1, Twist, Snail, and Vim are listed in Supplementary Table 1 . Relative gene expression was calculated from the qRT-PCR data using the 2 −ΔΔCT method.
5
Quantifying Gene Expression in Eucalyptus grandis
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Total RNA was extracted from E. grandis using the CTAB (cetyltrimethylammonium bromide) method (85 (link)), and first-strand cDNA synthesis was initiated using HiScript III reverse transcriptase (catalog number R323-01; Vazyme, Nanjing, China). Quantitative real-time PCRs were performed using ChamQ universal SYBR quantitative PCR (qPCR) master mix (Vazyme, Nanjing, China) in a 96-well real-time PCR system (Bio-Rad). All of the reactions were performed with three technical replicates of three biological replicates. The EgUBI3 genes from E. grandis and the RiEF1a gene from R. irregularis were used as the internal controls for normalization. The relative expression levels of the genes were computed by the 2−ΔΔCT method of relative quantification. A list of gene-specific primers used for qRT-PCR is given in Table S1 in the supplemental material.
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