Sybr exscript qrt pcr kit

To examine the expression level of the nutrition metabolism-regulation-related genes in hepatopancreas, four appetite-related genes were detected (Table S3). In detail, RNA extraction and cDNA synthesis were performed as described in a previous study [34 (link)]. qRT-PCR was carried out in an ABI 7300 Real-time Detection System (Applied Biosystems, Foster City, CA, USA) using the SYBR ExScript qRT-PCR Kit (Takara, Dalian, China) with β-actin as an internal reference [35 (link)]. The assay was conducted in three replicates, and the data were analyzed by the 2^−ΔΔCt method [36 (link)].

Tongue sole as described above were randomly divided into three groups and administered via intraperitoneal injection with 50 µl PBS containing 6.25 µM NKLP27 or P86P15. The control group was injected with 50 µl PBS. At 1 h, 12 h, and 24 h post-injection, tissues were collected and used for total RNA extraction with the RNAprep Tissue Kit (Tiangen, Beijing, China). One microgram of RNA was used for cDNA synthesis with the Superscript II reverse transcriptase (Invitrogen, Carlsbad, USA). The expression levels of interleukin (IL)-1β, IL-8, CsCCK1, CsCXCe1, toll-like receptor 9 (TLR9), myeloid differentiation factor 88 (Myd88), CsISG15, and CsG3BP were determined by qRT-PCR with primers reported previously [9] (link), [38] (link)–[40] (link). qRT-PCR was carried out in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) using the SYBR ExScript qRT-PCR Kit (Takara, Dalian, China) as described previously [41] (link). Melting curve analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and detected. The expression levels of the immune genes were analyzed using comparative threshold cycle method with beta-actin as the control.

Total RNA was extracted from cells or tissues with the RNAprep Tissue Kit (Tiangen, Beijing, China). One microgram of total RNA was used for cDNA synthesis with M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). qRT-PCR was carried out in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) using the SYBR ExScript qRT-PCR Kit (Takara, Dalian, China) as described previously [28] (link). Melting curve analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and detected. The expression level of the target gene was analyzed using comparative threshold cycle method with beta-actin (for samples without viral infection) or elongation factor-1-alpha (EF1 alpha) (for samples from virus-infected fish) as the internal reference [28] (link), [29] (link). All data are given in terms of mRNA levels relative to that of the internal reference and expressed as means plus or minus standard errors of the means (SE).

Total RNA was extracted from different treatment of the wheat seedlings leaves (0.2 g) by using PureLink^®RNA Mini Kit (Tiangen Biotechnology, Beijing, China). The quality of total RNA was quantified by the UV spectrophotometer. First-strand cDNA was synthesized by Revert Aid TM First Strand cDNA Synthesis Kit (Tiangen Biotechnology, Beijing, China). The qRT-PCR was performed in a 20 μl reaction volume tube using the SYBR ExScript qRT-PCR Kit (Takara, Dalian, China) according to the method described previously by Li et al. (2013) (link) and Qiu et al. (2013) (link). Specific primers for SOD, POD, and CAT genes, and the internal control tubulin gene were used to amplify amplicons specific for wheat seedlings. Specific primers were designed according to wheat EST sequences of candidate proteins available in NCBI (Qiu et al., 2014 (link); Zou et al., 2015 (link)), and the DNA sequences of specific primers are provided in Table 1. Melting curve analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and detected. Gene expression was counted and expressed relative to the expression levels of an internal reference gene actin in each sample using the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted from psoriatic HaCaT cells using TRIzol Reagent (Invitrogen) at 24 h after stimulation or transfection. The RNA was reversely transcribed to cDNA using a PrimeScript RT-polymerase kit (TaKaRa, Dalian, China) to the manufacturer’s protocol.
The expression levels of genes were detected using an SYBR ExScript qRT-PCR Kit (TaKaRa). GAPDH was used as the internal control. Gene-specific PCR primer pairs were synthesized by Sangon (Shanghai, China; Table 1). PCR amplification was conducted according to the following reaction conditions: 95 °C for 5 min; 38 cycles of 95 °C for 30 s, 60 °C for 40 s, and 72 °C for 45 s. The relative expression levels of genes were analyzed using the 2-^△△Ct methods.

Total RNA was extracted from different transfected cells using RNA Iso-plus reagent (Takara Bio, China) and then reverse transcribed into cDNA using PrimeScript RT Reagent Kit (Invitrogen) following the manufacturer's protocol. The primers in this study were designed using Primer 5.0 (Primer-E, Ltd., United Kingdom) as follows: miR-30c RT: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTGAG-3′; U6 RT 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGGAAC-3′. With a standard protocol provided by the manufacturer, qRT-PCR was performed in the ABI PRISM 7300 Fast Real-Time PCR System (Ambion, USA) using the SYBR ExScript qRT-PCR Kit (Takara, China), under the following conditions: 95°C for 5 min, 95°C for 10 s, and 40 cycles at 60°C for 30 s. Each reaction was performed in triplicate. The expression levels of miR-30c were normalized to U6. The results of RT-PCR are reported as 2^-ΔΔCt.