7500 qrt pcr system

Histochemical GUS staining was performed according to Jefferson et al. (1987). The stained tissues were rinsed and fixed in FAA (formalin‐acetic acid‐70% ethanol [1:1:18]) at 4 °C for 24 h. Then the stained materials were observed using stereo microscope. Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions (Invitrogen, http://www.invitrogen.com). First‐strand cDNA was synthesised from 3 μg of total RNA from each sample using M‐MLV reverse transcriptase (Promega, http://www.promega.com). Quantitative RT‐PCR (qRT‐PCR) was performed to monitor gene expression, and OsActin1 (LOC_Os03g50885) was used as a control. qRT‐PCR was carried out in the presence of the double‐strand DNA‐specific dye SYBR Green (Takara, China) and monitored in real time with a 7500 qRT‐PCR system (Applied Biosystems, Singapore).

We have used Lipofectamine™ RNAiMAX (Invitrogen) transfect miR-21-1 and miR-181b-2 mimics or miR-21-1 and miR-181b-2 inhibitors, NC mimic and NC inhibitor as negative control into MBrC cells. 24 hours after transfection, the MBrC cells were stimulated with LPS or poly(I:C) or SCRV, and then cells were collected with Trizol. Total RNA was isolated with Trizol reagent (Invitrogen) by following the Ultrapure RNA kit (CWBIO) instructions, and it was reverse transcribed into cDNA according to the HiScript II Q RT SuperMix (Vazyme Biotech) instructions. Then we designed specific RT primers to detect the expression of TRIF, TNF-α, Mx1 and IFN-2 genes, and β-actin was used as endogenous control to normalize the expression. The triple fluorescence intensity of each gene experimental group and control group was measured by Super Real PreMix Plus (Tian Gen) reagent and 7500 qRT-PCR system (Applied Biosystems, USA). At the end of the determination, the results of curve analysis were analyzed to determine the specificity of the target. For each sample, all amplification reactions were performed in triplicate. The sequences of all mRNA primers are listed in Supplementary Table 1.

Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions (Invitrogen). The cDNA was synthesized from 3 μg of total RNA from each sample using M-MLV reverse transcriptase (Promega). qRT-PCR was performed to monitor gene expression, carried out with SYBR Green (Takara), and monitored in real-time with a 7500 qRT-PCR system (Applied Biosystems, USA). The primers for gene expression analysis of OsAAP1 were 5'-GCACATTACAAGCCATTCAGCGTC-3′ and 5'-CTGACGAAACACTTGAGCACTC-3′. OsACTIN1 (LOC_Os03g50885) was used as an internal standard with the primers 5'-CGGTGTCATGGTCGGAAT-3′ and 5'-GCTCGTTGTAGAAGGTGT-3′.

Samples of fresh tissue (250–500 mg) were homogenized in 10× vol of cold potassium buffer, and levels of TNF-α protein were determined using enzyme-linked immunosorbent assay (Abcam, Shanghai, China). Total RNA from intestinal tissues was extracted with TRIzol^® Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The level of TNF-α mRNA was determined by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) using Power SYBR Green PCR Master Mix (TaKaRa Biotechnology, Shiga, Japan) and a 7500 qRT-PCR system (Applied Biosystems, Foster City, CA, USA). The housekeeping gene β-actin was used as the internal control. The primers specific for TNF-α and β-actin used for real-time qRT-PCR were as follows (forward and reverse, respectively): 5′-AAGAGGCACTCCCCCAAAAGAT-3′ and 5′-TCTGAGTGTGAGGGTCTGGGC-3′; 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′. Each reaction was performed in triplicate, and the fold change in the expression of each gene was calculated using the 2^−ΔΔCT method.

Total RNA was extracted using TRIzol reagent (Invitrogen, Beijing, China). First-strand cDNA was synthesized using oligo (dT) primers and MLV reverse transcriptase (TaKaRa Bio, Beijing, China). qRT-PCR was carried out using SYBR Green Premix (TaKaRa Bio) and monitored with the 7500 qRT-PCR system (Applied Biosystems, Foster City, CA, United States). To detection the expression level of two variants for OsNPF7.7, the primers sites for longer variant OsNPF7.7-1 were situated at the sequence of variant OsNPF7.7-1 its own, and the primers of OsNPF7.7-2 were designed at the common sequence of both two variants. Next, the actual expression amount of variant OsNPF7.7-2 was that both variants amount of OsNPF7.7 deduced the amount of OsNPF7.7-1. The primers for qRT-PCR were listed in Supplementary Table S1.

Using the 7500 qRT-PCR system (Applied Biosystems, South San Francisco, CA, USA), the relative expression levels of ScCIPKs under different exogenous stresses were assessed. The qRT-PCR primers of ScCIPKs were designed using Beacon Designer 8.12 software. The Cullin (CUL) [91 (link)] and Clathrin adaptor complex (CAC) [91 (link)] genes were used to normalize relative transcript levels. The qRT-PCR reaction system was prepared using the SYBR Green Master Mix (TaKaRa), following the manufacturer’s instructions. Each qRT-PCR was repeated thrice, and the reaction conditions were listed as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The qRT-PCR data was analyzed using the 2^-ΔΔCtmethod [92 (link)]. Statistical analysis was conducted using Data Processing System v9.50 software (China). Significance (p < 0.05) was calculated using one-way ANOVA, followed by Duncan’s new multiple range test. All of the primers used in qRT-PCR are listed in Supplementary Table S5.