Sybr green pcr premix

Total RNA from mouse small intestine was extracted with a TaKaRa MiniBEST Universal RNA extraction kit (Takara Bio Inc., Dalian, China). The concentration of RNA was quantified using a NanoReadfy Ultra-Micro UV-vis spectrophotometer (Hangzhou Suizeng Biotechnology Co., Hangzhou, China). A PrimeScript™ RT Master Mix kit (TaKaRa RR036A) was used for reverse transcription. A CFX-96 real-time quantitative PCR system (Bio-Rad, Hercules, CA, USA) was used to conduct qRT-PCR with SYBR Green PCR premix (TaKaRa RR820A). The primers for zonula occludens 1 (ZO-1, forward: 5′-GAGCTACGCTTGCCACACTGT-3′, reverse: 5′-TCGGATCTCCAGGAAGACACTT-3′) and occludin (forward: 5′-TGAAAGTCCACCTCCTTACAGA-3′, reverse: 5′-CCGGATAAAAAGAGAGTACGCTGG-3′) were used for the qRT-PCR. The 18S gene was used as internal reference (forward: 5′-AGTCCCTGCCCTTTGTACACA-3′, reverse: 5′-CGATCCCAGGGCCTCACTA-3′).

qPCR was used to determine the expression levels of aggrecan, COL2A1, TGFβ1, and RNA18S5N (internal control). Reactions were performed on a QuantStudio 7 Flex (Thermo Scientific) using a SYBR Green PCR premix (Takara). The qPCR program was set as follows: a preheating step at 95 °C for 60 s; followed by 40 cycles of heating (95 °C for 30 s), annealing (58 °C for 35 s), and extension (72 °C for 60 s); and a final extension (72 °C for 10 min). Relative mRNA expression levels were determined using the 2^−ΔΔCT method. The following primers were used for qPCR: TGFβ1 forward primer, 5′- CCGCAACAACGCAATCTA-3′; TGFβ1 reverse primer, 5′- TGCTTCCCGAATGTCTGA-3′; COL2A1 forward primer, 5′-GGAAGAGCGGAGACTACT-3′; COL2A1 reverse primer, 5′- TCCATGTTGCAGAAGACTT-3′; aggrecan forward primer, 5′- CTTCTGCCTCTGGAATAG-3′; aggrecan reverse primer, 5′-CACTGACATCCTCTACTC-3′; RNA18S5N forward primer, 5′- AGGCGCGCAAATTACCCAATCC-3′; and RNA18S5N reverse primer, 5′-GCCCTCCAATTGTTCCTCGTTAAG-3′.

Total RNA was extracted from frozen liver samples using the RNeasy Mini Kit (Qiagen Inc.) according to the manufacturer's protocol. RNA (2 μg) for each analyzed sample was reverse-transcribed in a final volume of 25 μl using M-MLV Reverse Transcriptase (Promega). Quantitative real-time PCR reaction was performed in a final volume of 20 μl containing 1 μl of cDNA (1 : 10 dilution) and 500 nM of primers (all primers were obtained from Qiagen) and SYBR Green PCR premix (Takara) according to the manufacturer's instructions. The expression levels of target genes were normalized to the expression of beta-actin and quantified based on the comparative cycle threshold Ct method (2^−ΔΔCt).

Peripheral blood was collected from patients and healthy individuals after fasting for 8 h. Then peripheral blood mononuclear cells (PBMCs) were isolated and kept in liquid nitrogen for further analysis. Total RNA was extracted from PBMCs using Trizol LS reagent (Invitrogen, Life Technologies, USA) and reverse transcribed into cDNA by M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Primers were shown in Table 2. Real-time PCR was performed with the SYBR GREEN PCR Premix (TaKaRa Biotechnology CO., Ltd., Dalian, P.R. China) following the manufacturer's protocols. Target genes and β-actin were amplified. The target genes were quantified by Ct value using 2^-ΔΔCt. Experiments were repeated for at least 3 times.Table 2

Primers used in this study

Primers	Sequences (5' to 3')
IL-4_F	TTTGCTGCCTCCAAGAACAC
IL-4_R	TTCCTGTCGAGCCGTTTCAG
IL-21_F	ACACAGACTAACATGCCCTTCA
IL-21_R	ACCGTGAGTAACTAAGAAGCAAATC
BCL-6_F	GGAAACCCAGTCAGAGTATTCG
BCL-6_R	CACATTTGTAGGGCTTTTCTCC
β-actin_F	TAGGCGGACTGTTACTGAGC
β-actin_R	TGCTCCAACCAACTGCTGTC
Blimp-1_F	TCCAGCACTGTGAGGTTTCA
Blimp-1_R	TCAAACTCAGCCTCTGTCCA

For qRT-PCR analysis, liver tissues were taken from parasitic lesions in E. multilocularis-infected mice and control mice as previously described (32 (link)). Total RNA was extracted from mouse liver tissue using TRIzol Reagent (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA; cat. no. K1622). Then, the cDNA was subjected to qRT-PCR by the SYBR Green PCR premix (TaKaRa, Dalian, China) in a thermocycler (iQ5 Bio-Rad, Hercules, CA) as previously described (34 (link)). Primer sequences for the genes analyzed are listed in Supporting Table S2. Gene expression was normalized to the housekeeping gene GAPDH. Relative mRNA expression was calculated using the 2^-ΔΔCt method.

Quantitative RT-PCR was used for analysis the mRNA levels of CD69, NKG2A, NKG2D, Ly49G2, Ly49I, Ly49D, Ly49H in whole-liver tissue at designated time-points after infection with E. multilocularis and normalized by comparison to GAPDH.
Total RNA, extracted from the liver using Trizol Reagent (Invitrogen, Canada), was reversed using M-MLV transcriptase according to manufactures’ instructions (Thermo, USA), then qRT-PCR (see primer information in Table 1) was implemented in thermocycler (iQ5 Bio-Rad, Canada) with SYBR Green PCR premix (TaKaRa, China) [29 (link)]. The results were calculated by the 2^−△△Ct method.
Table 1

Sequences of the qRT-PCR primers

Gene	Forward primer	Reverse primer
CD69	TGGTCCTCATCACGTCCTTAATAA	TCCAACTTCTCGTACAAGCCTG
NKG2A	TTCAGCACAGCCTTGTCCTC	CTTCTTTCCAGACCCAGGGC
NKG2D	CCAATGTTCGTTGTTCGAGTCC	GCACAATACTGGCTGAAACGTC
Ly49G2	TGCCACGATAACTGCAGCC	ATGGGTCTTTTGTGAACACCTG
Ly49I	GGAACAGTGAAACCAAGACGG	CTGTAATGCTGGCAGTTCGC
Ly49D	TTCAGGGTTGCAGAACGAGATGAG	AGGATCCCGAGAGCTATCACAATG
Ly49H	TGGGACAGTGAAACCAAGAGTG	GCTGTAATGCTGGCAGTTCG
GAPDH	CACTCACGGCAAATTCAACGGCAC	GACTCCACGACATACTCAGCAC

Total RNA was extracted using the RNAiso Plus reagent (TaKaRa) and PrimeScript RT kit (TaKaRa). Next, RNA was reverse transcribed to generate complementary DNA based on the procedure specified by the manufacturer. We used SYBR Green PCR Premix (TaKaRa) to perform qRT-PCR on the ABI7500 Fast Real-Time RCR System (Applied Biosystems, USA). Next, primers were designed using the PubMed database and synthesized by DynaScience Biotechnology (Guangzhou, China). All experiments were performed in triplicates. The results were normalized using GAPDH as an internal control. Finally, we used the 2-ΔΔCt method for the relative quantification of gene expression.