Sybr premix ex taq 2 2

The expression profiles of apoptosis and hypoxia-related genes in the hepatopancreas and gills were measured by qRT-PCR, which was implemented on the QuantStudio 6 Flex Real-time PCR System (Applied Biosystems, United States) using SYBR^® Premix ExTaq II (2×) (TaKaRa, Japan) following the protocols. All samples were run in triplicate and the M. japonicus elongation factor 1-α (EF1-α) was served as the reference gene (Table 1). Gene expression change was calculated by the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)). Expression data were shown with the mean ± standard deviation.

In this study, 11 PKS genes of upland cotton were quantitatively analysed by real-time fluorescence. Cotton bolls at 3 DAF, 6 DAF, 9 DAF, 12 DAF, 15 DAF, 18 DAF, 21 DAF were collected and RNA was extracted using the Tiangen (Beijing, China) plant RNA extraction kit. Reverse transcription was performed using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Tokyo, Japan) and each reaction used 1 µg of RNA. The specific primers for the PKS gene of upland cotton (Table S1) were designed using Beacon Designer 7 software and the internal reference gene used UBQ7 (Table S1). The qRT-PCR system consisted of 20 µL: 10 µL of SYBR^® Premix Ex Taq™ II (2 ×) (Takara), 2 µL of cDNA and 0.8 µL of GhPKS-F and GhPKS-R. The reaction procedure was 40 cycles of 50 °C for 2 min, 95 °C for 30 s, 95 °C for 5 s and 60 °C for 20 s, followed by 72 °C for 10 min; the experiment was repeated three times. Finally, we used 2^−ΔΔCt for the calculation of relative expression (Livak & Schmittgen, 2001 (link)).

The total RNA of Porphyromonas gingivalis was extracted by a Bacterial RNA Kit (Omega Bio-Tek, Norcross, GA, USA), and measured by Nanodrop2000 to determine the RNA concentration. Reverse transcription was performed by M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA) to generate cDNA. Amounts of mRNA transcripts were measured by the Roche LightCycler 480 real-time PCR detection system (Roche, Basel, Switzerland). The galE gene was used as the housekeeping amplicon [40 (link)]. Reactions were performed with 20 μL of a mixture containing 10 μL of SYBR Premix Ex Taq II (2×; Takara), 1.6 μL of each gene-specific primer, 3.4 μL of sterile distilled water and 5 μL of the cDNA template. The forward and reverse primer sequences are shown in Table 1 [40 (link),41 ]. Real-time PCR conditions included 30 s at 95°C; 10 s at 95°C, 20 s at 60°C and 15 s at 72°C for 40 cycles. Melting temperature curve analyses were used to validate the specificity of each primer pair. Data were analyzed using the comparative Ct method.

Total RNA was extracted using an E.Z.N.A.® Total RNA Kit II (Omega Bio-Tek, Norcross, GA) and reversely transcribed into cDNA using a ReverTra Ace-a Kit (Toyobo, Osaka, Japan). SYBR-Green real-time RT-PCR was performed using the Stratagene Mx3005P sequence detection system (Agilent Technologies, Santa Clara, CA) and SYBR Premix EX Taq II (2×) (Takara, Shiga, Japan). GAPDH mRNA was used to normalize the RNA inputs. All primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.

The soil dehydrogenase (DHA) activity was measured by monitoring the rate of the reduction of triphenyl tetrazolium chloride (TTC) to triphenyl formazan (TPF), as described by Oliveira et al.37 (link), with some modifications. TPF was detected using a spectrophotometer (UV-2550, Shimadzu) at 485 nm after a dark incubation for 24 h and expressed in μg TPF d·g⁻¹ of dry soil 24 h⁻¹.
The bacterial biomass was analysed using the real-time PCR of the 16S rRNA gene; this was performed with the ABI Prism 7000 Real-Time PCR Detection System (Applied Biosystems, USA) using SYBR Premix Ex Taq II (2×) and ROX Reference Dye (50×) (Takara, China). A standard curve was produced using genomic DNA extracted from E. coli. as a template to quantify the total number of bacterial 16S rRNA gene copies. The primers used for amplification of the 16S rRNA genes were 8F (5′-GAGAGTTTGATCCTGGCTCAG-3′) and 518R (5′-ATTACCGCGGCTGCTGG-3′). The conditions for the real-time PCR were 30 s at 95 °C and then 40 cycles of 95 °C for 15 s, 55 °C for 30 s, 72 °C for 45 s and 72 °C for 5 min.

The quantitative real-time PCR (qRT-PCR) was performed to investigate Malitaf mRNA expression levels in different tissues of healthy M. amblycephala. In addition, the mRNA expression pattern of Malitaf and Matnfα was determined in spleen after LPS stimulation by qRT-PCR. The β-actin and 18S rRNA was selected as an internal control to verify the successful reverse transcription and to calibrate the cDNA template in spatial and temporal expression analysis, respectively. The qRT-PCR was implemented using an ABI 7500 Real-time PCR system (Applied Biosystems, Foster, CA, USA) in a total volume of 20 µL, including 10 µL SYBR^® Premix Ex Taq™ II (2×) (TaKaRa, Dalian, China), 0.4 µL ROX Reference Dye II (50×), 0.4 µL of each primer (10 µmol∙L⁻¹), 2 µL 1:5 diluted cDNA, and 6.8 µL of PCR-grade water. The thermal profile was 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s, 60 °C for 34 s, and 72 °C for 30 s. Melting curve analysis of the amplified products was performed at the end of each PCR to confirm that a single PCR product was generated. The 2^−∆∆Ct method was used to analyze the expression levels of Malitaf and Matnfα genes [32 (link)]. The data obtained from three independent biological replicates were subjected to statistical analysis and the values represented the n-fold difference relative to the references (kidney and 0 h).

Total RNA was isolated with TRIzol reagent (Invitrogen) and cDNA was reverse transcribed using a PrimeScriptTM RT Reagent Kit (TaKaRa). SYBR-Green real-time PCR (RT-PCR) was performed with the ABI 7500 device (Applied Biosystems Inc.) using SYBR Premix Ex Taq II (2×) (TaKaRa). Cellular GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) was used to normalize the RNA inputs. Relative changes in gene expression were analyzed using the 2^−ΔΔCt method. All primers were synthesized by Shanghai GenePharma Co., Ltd., and are listed as following:
CD147: (forward) 5′-TCGCGCTGCTGGGCACC-3′ and (reverse) 5′-TGGCGCTGTCATTCAAGGA-3′; Cyclophilin A: (forward) 5′-CATGGTCAACCCCACGTGTTCTT-3′ and (reverse) 5′-TAGATGGACTTGCCACCAGTGCCAT-3′; IFNB1: (forward) 5′-AGGACAGGATGAACTTTGAC-3′ and (reverse) 5′-TGATAGACATTAGCCAGGAG-3′; IL-6: (forward) 5′-GACAGCCACTCACCTCTTCA-3′ and (reverse) 5′-AGTGCCTCTTTGCTGCTTTC-3′; ISG15: (forward) 5′-CACAGCCATGGGCTGGGACCTG-3′ and (reverse) 5′-GCACGCCGATCTTCTGGGTGA-3′; TNF-α: (forward) 5′-ATGAGCACTGAAAGCATGATCCGG-3′ and (reverse) 5′-CTACAACATGGGCTACAGGCTTGT-3′; GAPDH: (forward) 5′-AGCAATGCCTCCTGCACCACCAAC-3′ and (reverse) 5′-CCGGAGGGGCCATCCACAGTCT-3′.