Abi quantstudio 6 flex system

RNA extraction and RT-PCR were performed as described previously [30 (link)]. Total RNA was extracted with TRIZOL reagent following the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Potential contaminating genomic DNA was removed by RNase-free DNase treatment (Promega, Madison, WI, USA). cDNA was prepared with the MMLV Reverse Transcriptase (Takara, Japan). Relative quantitation used the ABI QuantStudio6Flex System (Thermo Fisher, USA) and determined by 2^−ΔΔct method. The primer sequences of real-time PCR are shown in the Supplemental Table 1. Each experiment was performed in triplicate.

N. benthamiana leaves infiltrated with PBS or the HrpP protein were collected from infiltrated areas at 6 hpi and ground in liquid nitrogen. Total RNA was isolated using a plant RNA 425 kit (catalog number R6827-02; Omega, USA) according to the manufacturer’s instructions. The FastKing RT kit (catalog number KR116-02; Tiangen, China) was used to remove genomic DNA and synthesize the first-strand cDNA. Tenfold-diluted solutions of the reaction products were used for the quantitative real-time PCR (qRT-PCR) assay. qRT-PCR was performed in a 12-μL volume with Bestar SYBR green quantitative PCR (qPCR) master mix (catalog number AG11701; Accurate Biology, China) on the ABI Quant Studio 6 Flex system (Thermo Fisher, USA).

The qPCR assay was performed to examine the quantity of gut bacteria using the universal bacteria primer (unibac), and the expression levels of two development regulation genes, ecdysone receptor (ecr) and ultraspiracle protein (usp); six nutrient metabolism-related genes, insulin-like peptides 1 (ilp1), insulin-like peptides 2 (ilp2), hexamerin 70a (hex70a), hexamerin 70b (hex70b), hexamerin 70c (hex70c), and hexamerin 110 (hex110); and four immunity genes, apidaecin, abaecin, hymenoptaecin, and defensin-1 were investigated. β-actin was used as the reference gene. The primers of these genes are shown in Table 1. qPCR was performed in a 10 μL reaction volume containing 5 μL qPCR SYBR Green Master Mix (Yeasen Biotech Co., Ltd., Shanghai, China), 0.25 μL each gene-specific primer (10 μM) (Table 1), 0.5 μL cDNA template, and 4 μL RNase-free water. The reactions were performed with an ABI QuantStudio 6 Flex System (Thermo Fisher Scientific, Waltham, MA, USA). Thermal cycling conditions were 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 65 s, and elongation at 72 °C for 20 s. The melting curve was obtained by raising the temperature from 65 to 95 °C for 10 s in 0.5 °C increments. Both technical and biological triplicates were performed in all experiments.

The qRT-PCR assays were performed to measure the expression levels of Snail, Cox-2, and Vegf-c. For 4T1, HuROE 4T1, and ΔHuR 4T1, the cells were treated with 1‰ DMSO or eltrombopag (5 μmol/L) for 12 h before RNA extraction. For the RAW264.7 cells, the model group was stimulated with LPS (100 ng/mL), and the eltrombopag groups were incubated with LPS (100 ng/mL) and eltrombopag (10 μmol/L or 20 μmol/L) for 12 h. The total cellular RNA was extracted using TRIzon Reagent (CWBIO, Nanjing, China) following the manufacturer’s instructions, and the RNA concentration was quantified with Nanodrop (DeNovix, USA). cDNAs were conducted using the HiFiScript gDNA Removal RT MasterMix kit (CWBIO, Nanjing, China), and quantitative PCR was performed with the ABI QuantStudio 6 Flex system (Thermo Fisher Scientific, USA) using MagicSYBR green master mix kit (CWBIO, Nanjing, China). The mRNA levels of selected genes were normalized to β-actin and analyzed with 2^−ΔΔCt method. The primers synthesized by Sangon Biotech (Shanghai, China) were as follows: Snail (F: 5′-CACAC GCTGC CTTGT GTCT3′ and R: 5′-GGTCA GCAAA AGCAC GGTT-3′), Cox-2 (F: 5′-GGTGC CTGGT CTGAT GATGT ATGC-3′ and R: 5′-GGATG CTCCT GCTTG AGTAT GTCG-3′), Vegf-c (F: 5′-CTACA GATGT GGGGG TTGCT-3′ and R: 5′-GATTG GCAAA ACTGA TTGTG AC-3′), and β-actin (F: 5′-GTGGG CCGCT CTAGG CACCA A-3′ and R: 5′-TGGCT TTAGG GTTCA GGGGG-3′).