Sybr premix ex taq 2 qpcr kit

Total RNA was extracted from wild-type and mutant fly testes by using Trizol reagent (Sangon), then cDNA was transcribed, according to the manufacturer’s protocol (Takara). Quantitative PCR was run on a CFX96 Touch ((BioRad) to measure total αTub67C mRNAs with rp49 as reference, according to the manufacturer’s protocol (SYBR Premix EX Taq™ II qPCR Kit, Takara). The following primers were used in this assay (Table S6).

The specification and validity of all primers were firstly tested via semiquantitative RT-PCR and the amplifications were triggered by Premix Taq^™ kits (TaKaRa, Dalian, China). The amplified products of each candidate gene were identified via electrophoresis on a 1.5% agarose gel. The validated PCR primers should account for a single specific amplified product with the correct size.
The quantitative real-time PCR reactions were performed with the Bio-Rad CFX96 Real-Time PCR system (Bio-Rad, USA) using a SYBR^® Premix Ex Taq^™II qPCR kit (TaKaRa, Dalian, China). The qRT-PCR reactions were conducted in mixtures constituted with 12.5 μl 2×SYBR Premix Ex TaqII (TaKaRa, Dalian, China), 0.5 μl of each amplification primer (20 μM), 10.5 μl ddH₂O, and 1 μl cDNA templates. Each group of reactions was accomplished with three repetitions and one negative control, in which 1 μl ddH₂O was used instead of cDNA as a template for PCR. After pre-denaturation at 94°C for 30 s, a 2 step-program of qRT-PCR was set as follows: denaturing at 94°C for 5 s, subsequently annealing at 56°C for 30 s, and repeated for 40 cycles. Finally, melting curves of qRT-PCR amplifications were performed to confirm the specificity of the primers again by heating up the products from 56°C to 95°C.

Total RNA isolation from longissimus dorsi and biceps femoris muscle tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) was performed according to the manufacturer’s instructions. Purified and quality extracted RNA was carried out, as previously described [16 (link)].
Quantitative real-time PCR (qRT-PCR) was performed in triplicate for each complementary DNA (cDNA) sample, using an SYBR^® Premix Ex Taq™ II qPCR kit (Takara Biotechnology Co. Ltd., Dalian, China) according to the manufacturer’s guidelines on an ABI7900HT real-time PCR system (Applied Biosystems, Forest City, CA, USA). Amplification was performed with the following conditions: denaturation for 10 min at 95 °C, followed by 40 PCR cycles of denaturation for 15 s at 95 °C, and annealing and extension for 60 s at 56–64 °C. Gene expression was normalized to GAPDH (internal reference) and presented as relative fold change compared with control (NP). All samples were measured in triplicate. The mRNA expression levels of target genes were calculated using the 2^−ΔΔCt method [21 (link)]. All PCR primers used in this study are listed in Table 2 [22 (link),23 (link)].

The tight junction (TJ) is formed by a protein complex and is widely used as a marker of intestinal integrity [27 (link)]. Mucosal tissues from the duodenum, jejunum and ileum were scraped with a glass slide, immediately placed in liquid nitrogen, and stored at −80 °C. Real-time PCR analysis for TJ mRNA expression was performed using a SYBR Premix Ex Taq II qPCR kit (TaKaRa Biotechnology, Dalian, China) on a LightCycler480 thermocycler (Roche, Mannheim, Germany). Gene-specific primers for the amplicons of zonula occludens (ZO)-1, occludin, and β-actin are listed in Table 1. Relative transcript levels were quantified by the 2^−∆∆CT method as described previously [28 (link)].Table 1

Primers used for real-time PCR analysis

Gene	Primers sequences	Size in base pairs
ZO-1	F: 5′-TGAGTTTGATAGTGGCGTTG-3′	298
	R: 5′-TGGGAGGATGCTGTTGTC-3′
Occludin	F: 5′-CTAGTCGGGTTCGTTTCC-3′	167
	R: 5′-GACTGATTGCCTAGAGTGT-3′
β-actin	F: 5′-TGCGGGACATCAAGGAGAAGC-3′	273
	R: 5′-ACAGCACCGTGTTGGCGTAGAG-3′

Total RNA was independently extracted from Drosophila ovaries with different genotypes (wild-type and cycB3 mutants), using the Trizol Reagent (Sangon, Shanghai, China), and cDNA was synthesized, according to the manufacturer’s protocol (PrimeScript RT reagent Kit with gDNA Eraser, Takara, Dalian, China). Quantitative PCR was run on a CFX96 Touch (BioRad, Hercules, CA, USA) to measure total cycB3 mRNAs, with rp49 as a reference, according to the manufacturer’s protocol (SYBR Premix EX Taq™ II qPCR Kit, Takara, Dalian, China). The following primers were used in this assay (Table 3):