Takara sybr premix ex taq 2

RNA was extracted from the erythrocytes on 14 d.p.i.. The quantitative expression analysis of AvBDs and LEAP-2 genes were performed with real-time RT-PCR using a TaKaRa SYBR Premix Ex Taq™II (Takara Bio Inc.). The total volume of real-time PCR mixture contained 6 μL 1× SYBR Premix Ex TaqII, 0.1 μL ROX dye II, 1.5 pmol each of forward and reverse primers, 1 μL of diluted cDNA, and 2.6 μL of double distilled H₂O. The reaction mixture was placed into MicroAmp® Optical 8-Cap Strip (Applied Biosystems), and real-time PCR was performed following user instructions of the QuantStudio™ 6 (Applied Biosystems). Briefly, each reaction involved a pre-incubation at 95 °C for 3 min, followed by 42 cycles of 95 °C for 30 s, 55 °C - 57 °C (T_A as per primer) for 30 s, and extension at 72 °C for 10 s. The expression level of AvBDs and LEAP-2 were calculated relative to that of the housekeeping gene 18S rRNA using the QuantStudio™ 6 Flex Real-Time PCR System Software (Applied Biosystems, USA).

For each sample, 1 μg total RNA was used for cDNA synthesis using the PrimeScript Kit (TaKaRa Biotechnology, Dalian, China). TaKaRa SYBR Premix Ex Taq II (TaKaRa Biotechnology, Dalian, China) was used for qPCR. qPCR was performed on the Roche Light Cycler 96 System (Roche, Swiss). Each sample contains three independent biological replicates. The information of the primers used in the qPCR analysis was listed in S1 Table.

Total RNA was extracted using TRIzol reagents (Invitrogen) according to the manufacturer’s instructions. For mRNA and miRNA analysis, cDNAs were synthesized using the PrimeScript RT reagent kit (Takara). Then, qPCRs were performed by using the Takara SYBR Premix Ex Taq II (Takara) and the 7500 Real-Time PCR System (Applied Biosystems). The primer sequences for Snail [12 (link)], E-cadherin [12 (link)], ZO-1 [12 (link)], N-cadherin [12 (link)], Vimentin [12 (link)], CD44 [13 (link)], CD133 [12 (link)] and GAPDH [12 (link)] have been previously reported. For miRNA analysis, qPCRs were performed using the NCode miRNA qRT-PCR analysis (Invitrogen, CA, USA). Forward primer is the exact sequence of the mature miR-137 and miR-34a. The mRNA and miRNA expression data were normalized to GAPDH and U6, respectively. Results were represented as the fold change relative to respective controls.

Total RNA was extracted from the strawberry using Quick RNA Isolation Kit (Hua Yue Yang, Beijing China). The cDNA was reverse transcribed according to HifairTM II 1st Strand cDNA Synthesis SuperMix Kit for qRT-PCR (gDNA digester plus) (Yeasen, Shanghai, China). Specific primers were designed using online software Primer 3 Plus (http://primer3.ut.ee/; Supplementary Table 1). For different transcripts, NCBI Gene (https://www.ncbi.nlm.nih.gov) was used for comparing the difference of sequences between different transcripts, and then designed specific primers using the different part of the transcript sequence. TaKaRa SYBR Premix Ex Taq™ II (Takara, Dalian, China) was used to perform qPCR on ABI prism 7900 Real-Time PCR system (Applied Biosystems, USA). The thermal cycles were set as follows: 95°C for 2 min for pre-incubation, 40 cycles of 95°C for 10 s and 58°C for 30 s for amplification. The relative gene expression levels were calculated by the 2^−ΔΔCt method. The strawberry gene Actin was used as the reference.

The differentially expressed genes that were overexpressed in the BP category of “response to salt and osmotic stress”, were searched through BLAST against nucleotide databases and were assigned to their homologous genes. Among them, ten genes were randomly selected as representatives to validate the data generated through RNA-Seq. The primer pairs for qRT PCR were designed by Primer3 [51 (link),52 (link)] using the unigene sequences obtained in this study. The qRT-PCR was carried out using aliquots of the same RNA samples that were used for RNA sequencing using a Thermocycler BioRad-USA qPCR machine. Two micrograms (2 μg) of total RNA were used for cDNA synthesis by Prime Script II reverse transcriptase (TAKARA- Bio Inc. Japan) using an oligo(dT) primer. ACT2 gene was used as internal control for normalization. The cDNA was diluted 5 to 10-fold, and the qPCR reactions were carried out with two technical replicates, using Takara SYBR Premix Ex Taq II (Takara Bio Inc. Japan) and incubated at 95 °C for 3 min followed by 40 cycles of 95 °C for 15 s, 58 °C for 15 s and 72 °C for 15 s. The primer sequences for the unigenes are provided in Table S33. PCR specificity was evaluated using melting curve analysis, and the expression levels were calculated using the 2^−∆∆Ct method [53 (link)]. Data were analyzed and plotted using Microsoft Excel 2010.

A quantitative PCR (qPCR) assay was performed because of its high sensitivity and for detection of minor clade differences. We targeted clades A, C and D based on published reports [16 , 17 ] and preliminary trials, as only those clades were observed in all of the studied giant clams. To detect and quantify these clades, primer sets specific for the Symbiodinium clades (SymA28S-1F and SymA28S-1R for clade A, SymC28S-1F and SymC28S-1R for clade C, and SymD28S-1F and SymD28S-1R for clade D) were used [24 ]. qPCR was conducted using the prepared DNA extracts and each clade-specific PCR primer according to the protocol found in [24 ]. Briefly, mixtures of 1 ng μl^-1 DNA, 0.4 pmol μl^-1 of each forward and reverse primer, and the recommended volume of TaKaRa SYBR Premix Ex Taq II (Takara Bio Inc., Shiga, Japan), which included a ROX reference dye (as a passive reference), were analyzed in a StepOne (Applied Biosystems, Foster City, CA, USA) under the following PCR settings: 1 cycle at 95°C (30 s) and 40 cycles at 95°C (5 s) and 60°C (31 s).

The RNA were extracted from plant leaves by using a Spectrum Plant Total RNA kit (Sigma St. Louis, MO, USA) following the operating instructions.
The primer design and synthetic were accomplished in Sangon Biotech in Shanghai. The Reverse Transcriptase M-MLV (RNase H-) kit (TaKaRa Biotechnology. Lanzhou, China) was used to synthesize cDNA. Light Cycler^® 96 Real-Time PCR System (Roche, Basel, Switzerland) was used to perform qRT-PCR of VvEXO70s. The gene primers (Table S2) were designed in Sangon and used for PCR amplification, among which GAPDH gene (GenBank accession no. CB973647) was internal control genes. The amplification volume was 25 µL containing 1 µL forward primer, 1 µL reverse primer, 1.5 µL cDNA, 9 µL ddH₂O and 12.5 µL TaKaRa SYBR Premix Ex Taq. II (TaKaRa Biotechnology. Lanzhou, China). The response procedures was: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. For melting curve analysis, a program including 95 °C for 15 s, followed by a constant increase from 60 °C to 95 °C, was included following the PCR cycles. Each treatment was run three biological replicates. The 2^−ΔΔCT method was used to calculate the relative expression levels of genes (Udvardi, Czechowski & Scheible, 2008 (link)).