Tb green premix ex taq reagent

Total RNA was isolated using TRIzol reagent (Invitrogen). Gene expression was assayed using TB Green Premix Ex Taq reagent (TAKARA) according to the manufacturer’s protocol and normalized to GAPDH mRNA levels. All experiments were assayed on an ABI ViiA 7 Real-Time PCR System (Applied Biosystems). All primers for SYBR Green real-time qPCR were synthesized by Sangon Biotech. The primers are as follows: mmu-IFN-α-forward (5′-3′), ATG GCT AGG CCC TTT GCT TTC; mmu-IFN-α-reverse, CTG TGT ACC AGA GGG TGT AGT T; mmu-CXCL10-forward, GAC GGT CCG CTG CAA CTG; mmu-CXCL10-reverse, GCT TCC CTA TGG CCC TCA TT; mmu-IL-6-forward, CCA GTT GCC TTC TTG GGA CT; mmu-IL-6-reverse, GTC TCC TCT CCG GAC TTG TG; mmu-GAPDH-forward, CAG AAC ATC ATC CCT GCA TC; and mmu-GAPDH-reverse, CTG CTT CAC CAC CTT CTT GA.

RNeasy Mini Kit (Qiagen, Hilden, Germany) was used to extract the total RNA from the colon samples according to the product manual. The extracted total RNA was immediately reverse-transcribed to cDNA, and the cDNA was then stored at −80°C for further use. mRNA relative levels were measured in duplicate with TB Green Premix Ex Taq Reagent (TAKARA, Kusatsu, Japan) using an Applied Biosystems VIIA7 Real-time PCR system (Applied Biosystems, Waltham, MA, United States) (refer to Supplementary Table 1 for primer information). The relative mRNA expression levels of targeted genes was normalized by GAPDH expression.

Total RNA was extracted from rat hepatocytes using TRIzol reagent (TKR-9108, Takara, Japan) according to manufacturer's instructions. A reverse transcription reaction was then performed using a reverse transcription reagent with gDNA Eraser (TKR-RR047A, Takara, Japan) to reverse transcribe total RNA into cDNA, and finally using TB Green® Premix Ex Taq™ reagent (RR420A, Takara, Japan). Japan) RT-qPCR was performed on Step One Plus (Applied Biosystems, Carlsbad, CA, USA). β-actin is used as an internal reference gene to correct relative gene expression. The relative expression was calculated by 2^-ΔΔCt. The primer sequences used in the study are supplemented in Supplementary Table S1.

Total RNA was extracted and purified from K562 cells using RNAiso Reagent (Takara, Japan) according to the manufacturer’s instructions. 1 μg of total RNA was reverse transcribed using the PrimeScript RT Reagent Kit (Takara, Japan) to detect relative mRNAs. Real-time PCR was performed in triplicates on an Applied Biosystem7900 quantitative PCR system (Applied Biosystems, USA) using TB Green Premix Ex Taq reagent (Takara, Japan). The Ct values obtained from different samples were compared using the 2^−ΔCt method. GAPDH served as the internal reference gene.

The total RNA of pools (50 L. striatellus) of second-to-fifth instar nymphs, female and male adults and various organs (central nervous system, salivary gland, intestine, ovary and testis) dissected with tweezers under an optical microscope from 100 L. striatellus adults were extracted by RNAiso plus (Takara), according to the manufacturer’s instructions. The cDNA was reverse-transcribed from 1 μg extracted total RNA with PrimeScript™ RT reagent kit with gDNA Eraser (Takara), following the manufacturer’s instructions. RT-qPCR was conducted by using IQ™ 5 multicolor real-time PCR detection system (BIO-RAD) with TB Green Premix Ex Taq reagent (Takara). The relative gene expression was normalized to an internal control gene, alpha tubulin (primers listed in Table S1), as we described previously [27 (link)], and calculated by 2^−ΔΔCT (cycle threshold) method. Each experiment contained three independent biological and three technical replications.

Total RNA was extracted from the colon tissues by the RNeasy plus Minikit (Qiagen, Hilden, Germany) and was immediately reverse-transcribed to cDNA and stored at −80 °C until further procedures. mRNA relative levels were measured in duplicate with TB Green Premix Ex Taq Reagent (TAKARA, Kusatsu, Japan) using an Applied Biosystems VIIA7 Real-time PCR system (Applied Biosystems, Waltham, MA, USA), and threshold cycle (^ΔΔCT) was finally calculated to analyze the significant difference between groups. Primers are displayed in Table S2.

Total RNA in small intestine and liver tissues was extracted by applying TRIzol regent (Tiangen Biotech Co., Ltd., Beijing, China). EasyScript Plus cDNA synthesis kit (Takara Bio, Shiga, Japan) was used to synthesize cDNA according to the manufacturer’s protocol. qRT-PCR assay was carried out in a reaction system of 25 µl by applying TB Green Premix Ex Taq reagent (Takara Bio, Shiga, Japan) with the fluorescent PCR instrument (CFX96; Bio-Rad Laboratories Inc., Hercules, CA, USA). The amplification reaction was undertaken as follows: at 95 °C for half a minute, followed by 40 cycles at 95 °C for 5 s and at 60 °C for half a minute. Besides, β-actin, as a house-keeping gene, was used as a reference. Relative gene expression levels were determined using the 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }{\text{C}}_{\text{t}}}$ method. The primer sequences for the target genes are listed in Table S2.