Realmastermix sybr green kit

To validate the RNA-seq results, a subset of DEGs were verified by qPCR. An independent set of samples was harvested at 48 h after waterlogging, and a corresponding control was used for the expression analysis. Sequences for each gene from the 9930 genome database (http://www.icugi.org/cgi-bin/ICuGI/index.cgi) were used to design primers with the online Primer 3 tool (http://bioinfo.ut.ee/primer3-0.4.0/; Additional file 1: Table S1). The expression levels of 15 cucumber genes were tested using the RealMasterMix (SYBR Green) kit (Tiangen, China) following the manufacturer’s protocol with an iQ™ 5 Multicolor real-time PCR detection system (Bio-RAD, USA). To verify the presence of specific products, melting curve analyses of amplification products were executed following each PCR reaction.

To identify the potential function of the OPH-like genes, total RNA of CF-BD cultivated for 20 h in the BHI medium containing 0, 12.5, 25, and 50 mg trichlorphon were extracted to analyze the expression of the OPH-like genes. Complementary DNA (cDNA) was reverse-transcribed from 2 μg total RNA using MMLV reverse transcriptase (Promega). The recA gene was used as the reference gene [66 (link)]. Real-time quantitative PCR amplification was performed using Mx3000P spectrofluorometric thermal cycler (Agilent Technologies, Santa Clara, CA, USA) and Real Master Mix (SYBR Green) kit (Tiangen), starting with a 2 min incubation at 95 °C, followed by 40 cycles of 95 °C, 20 s; 55 °C, 1 min; and 72 °C, 30 s. Primer information for the genes was described in Additional file 3: Table S3.

Total RNAs were extracted from insects and S2 cells using TRIzol reagent (Invitrogen), and first-strand cDNAs were reverse transcribed using FastQuant RT kit with gDNase (Tiangen). qRT-PCR was performed using a RealMasterMix SYBR Green kit (Tiangen) with a LightCycler 96 system (Roche), initiated at 95°C for 15 min, and followed by 40 cycles of 95°C for 10 s, 58°C for 20 s, and 72°C for 30 s. Relative expression levels were calculated using 2^−ΔΔCt method, normalized by ribosomal protein 49 (Rp49). Primers for qRT-PCR are listed in Table S3 (Additional file 1).

Total RNA was isolated from leaves using RNAiso Plus (Takara, Dalian, China). Dried RNA samples were dissolved in DEPC-water to 1 μg/μL using a BioPhotometer Plus spectrophotometer (Eppendorf, Hamburg, Germany). RNA was reverse-transcribed using a Takara PrimeScript^® RT reagent kit with a gDNA eraser according to the manufacturer’s specifcations. qRT-PCR was performed using a RealMasterMix (SYBR Green) kit (Tiangen, Beijing, China) according to the manufacturer’s specifications. SYBR Green PCR cycling was performed using an iQ™ 5 multicolour real-time PCR detection system (Bio-Rad, Hercules, CA) with 20 μL samples. PCR primers were designed using Primer Premier 5.0 to avoid conserved regions. Primer sequences are listed in Supplementary Table 1. Wheat TaEF-α (No. Q03033) was used as internal reference gene. The relative quantities were calculated using the 2^−ΔΔCt method^{43 (link),44 (link)}. Each treatment included three replications, and each replication included two technical replications.

Total RNA of the cells after stimulation was extracted using Trizol Kit (Tiangen Biotech (Beijing) Co., Ltd). RNA purity and content were determined with UV/visible spectrophotometer. Synthesis of cDNA was conducted with TaKaRa reverse transcription kit (PrimeScriptII1st Strand cDNA Synthesis Kit) according to the manufacturer's instructions. After reverse transcription, the resulting materials were used for PCR amplification using gene-specific primer pairs and RealMasterMix (SYBR Green) kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China). α-SMA, K_ca3.1, FSP-1 and GAPDH mRNA were determined quantitatively by using Thermal Cyeler Dice Real Time System (TP800, Takara, Japan) and GAPDH as intra-contrast gene or internal control. All steps were performed on ice.
The sequences of α-SMA, Kca3.1, FSP-1 and GAPDH are shown in Table 1 and the amplification conditions for real-time PCR were: initial denaturation (95°C, 3 min), 40 cycles of denaturation (95°C, 10 sec), annealing (60.5°C, 10 sec), extension (72°C, 10 sec), and then a final extension (72°C, 10 min).

Total RNA was extracted from fat bodies using Trizol reagent (Invitrogen). cDNA was reverse-transcribed from DNase-treated total RNA (2 µg) by MMLV reverse transcriptase (Promega). qRT-PCR was conducted using an MX3000P spectrofluorometric thermal cycler (Stratagene) and RealMasterMix (SYBR Green) kit (Tiangen), initiated at 95°C for 2 min, followed by 40 cycles of 95°C for 20 s, 58°C for 20 s, and 68°C for 20 s. Melting curve analysis was performed to confirm the specificity of amplification. Relative mRNA levels were normalized by β-actin, and the 2^−ΔΔCt method was used to analyze relative gene expression levels. Primers used for qRT-PCR are listed in Table S2.