At day 2 post-transfection (pt) with pUC-HB-Ce and pcDNA-F-PUF60 or pcDNA-F-PUF60-D1, aliquots of the cells were harvested (designated as 0 h) and the other cells were treated with actinomycin D (5 µg/ml), followed by further culture for 6 or 12 h. At each time point, total RNA was extracted from transfected cells with TRI Reagent and the HBV 3.5 kb RNA level was determined by RT-qPCR using the SYBR qPCR Mix kit (Toyobo).
Sybr qpcr mix kit
Manufactured by Toyobo
Sourced in Japan
The SYBR qPCR Mix kit is a laboratory reagent designed for quantitative real-time polymerase chain reaction (qPCR) analysis. It contains all the necessary components, including a specialized DNA polymerase, buffer, and SYBR Green dye, to facilitate the detection and quantification of DNA targets in a qPCR workflow.
Automatically generated - may contain errors
Lab products found in correlation
Revertra acer kit, by Toyobo (4 mentions)
Rneasy plus mini kit, by Qiagen (3 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution try, by Solarbio (1 mentions)
Methotrexate mtx, by Yuanye Bio-Technology (1 mentions)
Tnf α cytokines, by Thermo Fisher Scientific (1 mentions)
Revertra ace qpcr rt master mix with gdna remover kit, by Toyobo (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Chitosan, by Sinopharm (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Solarbio (1 mentions)
Penicillin streptomycin mixture, by Solarbio (1 mentions)
Aldara imq cream, by 3M (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Dnase 1, by Toyobo (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Dnase 1 2270a, by Takara Bio (1 mentions)
Yeast total rna extraction kit, by Sangon (1 mentions)
Rnase free dnase 1, by Toyobo (1 mentions)
Icycler iq, by Bio-Rad (1 mentions)
11 protocols using sybr qpcr mix kit
1
Determining HBV RNA Stability
2
Quantification of gene expression in rice
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To investigate the expression of pyrimidine synthesis genes, young leaves (the youngest fully expanded leaves) and old leaves (the fourth leaf from the top) were collected from the six-week-old WT and mutant. To investigate the expression levels of plastid genes, young leaves were collected from the six-week-old WT and mutant, or leaves were collected from the one-week-old WT, complementation lines, and the mutant. To examine the expression of UMPK or cold-inducible genes under normal and abiotic stresses conditions, young leaves were collected from the four-week-old WT and mutant, or leaves were collected from about one-week-old WT plants, complementation lines, and the mutant. Total RNA was extracted from these rice tissues using an RNeasy Plus Mini Kit (QIAGEN, German). First-strand cDNA was synthesized using a ReverTra AceR kit (Toyobo, Japan). Quantitative real-time PCR was performed on an Applied Biosystems 7500 using SYBR qPCR Mix kit (Toyobo, Japan). Actin1 was used as the endogenous control. Data were analyzed according to the 2−ΔΔCt method [53 (link)]. Three independent biological replicates, each consisting of five plants for tissue samples, and three technical replicates per biological replicate were used. Primers are listed in Table S1 .
3
DNA Extraction, PCR, and Expression Analysis in Rice
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For population development and QTL mapping, total DNA was extracted using 2 cm-long leaf sample following the method of Zheng et al. [34 ]. PCR amplification was performed according to Chen et al. [35 (link)]. The products were visualized on 6% non-denaturing polyacrylamide gels using silver staining or on 2% agarose gels using Gelred staining. Three DNA markers were used, including functional marker Si9337 for Hd1, functional marker Se9153 and closely linked marker RM5436 for Ghd7 [10 (link),17 (link)].
For expression analysis, penultimate leaves of rice lines in the R1-NIL population were harvested at 7:00 am in 17HZ and 9:00 am in 17LS, 2 h after sunrise. Total RNA was extracted using RNeasy Plus Mini Kit (QIAGEN, Hilden, German). First-strand cDNA was synthesized using ReverTra AceR Kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed on Applied Biosystems 7500 using SYBR qPCR Mix Kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Actin1 was used as the endogenous control. The data were analyzed according to the 2-ΔCt method. Three biological replicates and three technical replicates were used. The primers were selected from previous studies [10 (link),20 (link),36 (link)].
For expression analysis, penultimate leaves of rice lines in the R1-NIL population were harvested at 7:00 am in 17HZ and 9:00 am in 17LS, 2 h after sunrise. Total RNA was extracted using RNeasy Plus Mini Kit (QIAGEN, Hilden, German). First-strand cDNA was synthesized using ReverTra AceR Kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed on Applied Biosystems 7500 using SYBR qPCR Mix Kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Actin1 was used as the endogenous control. The data were analyzed according to the 2-ΔCt method. Three biological replicates and three technical replicates were used. The primers were selected from previous studies [10 (link),20 (link),36 (link)].
4
Transcriptomic Analysis of Rice Panicle
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Young panicles in three periods (1–5 cm, 6–10 cm and 11–15 cm) were collected from NIL-TQ, NIL-IR, TQ and S2. Young panicles in one period (11–15 cm) were collected from TQ, ZY179, S1, S2, D1-1 and D1-2. Total RNA was extracted using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). The first-strand complementary DNA was synthesized using ReverTra AceR Kit (Toyobo, Osaka, Japan). qRT-PCR was performed on Applied Biosystems 7500 using SYBR qPCR Mix Kit (Toyobo, Osaka, Japan). Actin was used as the endogenous control. Relative gene expression was analyzed according to the 2−ΔΔCt method [49 (link)]. Three biological replicates were performed for each line, and three technical replicates were performed for each sample. The qRT-PCR primers are listed in Table S7 .
5
Anti-inflammatory Mechanism of Gentiopicroside in Psoriasis
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Gentiopicroside was purchased from Beijing Zhongke Quality Inspection Biotechnology Co., Ltd. (Beijing, China). Chitosan was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Aldara IMQ Cream was obtained from 3M Health Care Limited (Shanghai, China). TNF-α cytokines were purchased from PeproTech (Cranbury, NJ, USA). BALB/c mice were purchased from Beijing Weitong Lihua Laboratory Animal Co., Ltd. (Beijing, China). HaCaT cells were purchased from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China).
Minimum essential medium (MEM) and fetal bovine serum (FBS) were purchased from Gibco (Waltham, MA, USA). Penicillin-streptomycin mixture, annexin V-FITC/PI apoptosis detection kit, and trypsin-EDTA solution (TRY) were obtained from Solarbio Life Science (Beijing, China). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Beijing Aokesi Technology Co., Ltd. (Beijing, China) and methotrexate (MTX) was purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). ReverTra Ace®qPCR RT master mix with gDNA remover kit and SYBR®qPCR mix kit were purchased from TOYOBO (Tokyo, Japan).
Minimum essential medium (MEM) and fetal bovine serum (FBS) were purchased from Gibco (Waltham, MA, USA). Penicillin-streptomycin mixture, annexin V-FITC/PI apoptosis detection kit, and trypsin-EDTA solution (TRY) were obtained from Solarbio Life Science (Beijing, China). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Beijing Aokesi Technology Co., Ltd. (Beijing, China) and methotrexate (MTX) was purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). ReverTra Ace®qPCR RT master mix with gDNA remover kit and SYBR®qPCR mix kit were purchased from TOYOBO (Tokyo, Japan).
6
Fractalkine-Induced Inflammatory Response
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Cells were harvested after treatment with or without fractalkine for 6 h, in the absence or presence of 2-APB, and after fractalkine (10 nM) treatment for 0, 6, 12, or 24 h. Total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA). Mice in the fractalkine group were treated as described for the ELISA assay. Reverse transcription was performed according to standard protocols using a reverse transcriptase assay kit (Invitrogen). Expression levels of target genes were determined from cDNA samples according to standard protocols using a SYBR qPCR mix kit (Toyobo, Osaka, Japan). PCR cycling protocols were as follows: initial denaturation, 95°C for 1 min; cycling, 95°C for 15 s, 58°C for 10 s, and 72°C for 45 s; for 40 cycles. The following primers were used for mRNA detection: IL-1β forward, 5′-TTTGAAGTTGACGGACCCC-3′; reverse, 5′-GTGCTGCTGCGAGATTTGA-3′; TNF-α forward, 5′-CGGGCAGGTCTACTTTGGAG-3′, reverse, 5′-CAGGTC ACTGTCCCAGCATC-3′; actin forward, 5′-CCGTGAAAAG ATGACCCAG-3′, reverse, 5′-TAGCCACGCTCGGTCAGG-3′. For relative comparison of each gene, realtime PCR data were evaluated using the equation 2−ΔCt test/2−ΔCt control.
7
Quantifying HBV DNA and RNA in Cell Culture
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Quantification of HBV DNA and RNA was performed as previously described [15 ]. The HBV DNA in the culture supernatant collected from the transfected cells was treated with PNE solution (8.45% PEG, 0.445 mole NaCl and 13 mmol EDTA) for 1 h on ice. The pellets were incubated with DNase I (TAKARA, Shiga, Japan) and RNase (TaKaRa) for 1 h at 37°C. The pellets were then treated with proteinase K for 12 h at 56°C, and HBV DNA was separated by phenol/chloroform extraction and ethanol precipitation. HBV DNA copies were determined by qPCR. For quantification of HBV 3.5 kb pgRNA, total RNA was extracted from HBV-transfected cells using TRI reagent (Molecular Research Center, Cincinnati, OH, USA). After treatment with DNase I and RNase inhibitor, cDNA templates were synthesized and HBV RNAs were quantified by qPCR using the SYBR qPCR Mix kit (Toyobo, Osaka, Japan) using 5′-TCCCTCGCCTCGCAGACG-3′ and 5′-GTTTCCCACCTTATGAGTC-3′ for unspliced 3.5 kb RNA, β-actin mRNA primers (5′- TTCTACAATGAGCTGCGTGTG-3′ and 5′-GGGGTGTTGAAGGTCTCAAA-3′). For semi-quantitative RT-PCR, cDNA templates were amplified with primers as previously reported [15 ].
8
Tissue-specific Expression of LUC7L3, SMC5, and SMC6
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Expression of LUC7L3 mRNA in various tissue was examined by using the multiple tissue cDNA (MTC) human panels I and II (Clontech laboratories, CA, USA). The panels contained a set of normalized single-strand cDNAs, produced from poly(A)+ RNA from various normal human tissues. qPCRs were performed using the SYBR qPCR Mix kit (Toyobo, Osaka, Japan). Expression of LUC7L3 mRNA was standardized by that of RNA polymerase II (RPII) gene. The primers used were 5′-TCAAGCCGAACATCAGACAG-3′ and 5′-GCTTCTGCTTCTTCGTCGAT-3′ for LUC7L3 and 5′-GCACCACGTCCAATGACAT-3′ and 5′-GTGCGGCTGCTTCCATAA-3′ for RPII. For RT-qPCR to determine mRNAs of SMC5 and SMC6, total cellular RNAs isolated by TRI reagent were transcribed using SuperScript VILO cDNA Synthesis Kit. Aliquots of cDNAs were subjected to 45 cycles of PCR amplification using THUNDERBIRD SYBR qPCR mix. The primers used were 5′-AGAAGCAAGATGTTATAGAAAGGAAAG-3′ and 5′-TCCTCTGTCGGTCAAGCTCT-3′ for SMC5 and 5′-TGCATCAATTCTGGACAAAGA-3′ and 5′- TGCTTCTTGGTACTGCCTCA-3′ for SMC6. The RNA expression data were normalized to levels of reference gene GAPDH using the comparative threshold cycle method.
9
Quantification of HBV DNA and pgRNA
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HBV DNA in transfected cells culture supernatant was treated with PNE solution (8.45% polyethylene glycol (PEG), 0.445 mol NaCl, 13 mmol EDTA) in ice for 1 h, and incubated with Dnase I (2270A, TAKARA, Shiga, Japan) and Rnase (2158, TAKARA , Shiga, Japan) at 37 °C for 1 h. Microspheres were treated with proteinase K at 56 °C for 12 h, and HBV DNA was isolated by phenol/chloroform extraction-ethanol precipitation method. HBV DNA copy number was determined by quantitative polymerase chain reaction (qPCR). To quantify HBV 3.5 kb pgRNA, total RNA was extracted from HBV transfected cells using total RNA extractor (TRI) reagent (TR 118, Molecular Research Center, Cincinnati, OH, USA). After treatment, using Dnase I and Rnase inhibitor, cDNA template was synthesized. Unspliced 3.5 kb RNA was quantified by qPCR using Synergy BrandsSynergy Brands (SYBR) qPCR Mix kit (QKD-201T, Toyobo, Osaka, Japan). The thermal cycling conditions comprised 1 min at 95 °C, followed by 40 cycles at 95 °C for 15 sec and 60 °C for 30 sec. Primer sequences used in this study were: 5′-TCCCTCGCCTCGCAGACG-3′ and 5′-GTTTCCCACCTTATGAGTC-3′ for unspliced 3.5 kb RNA, and 5′-TTCTACAATGAGCTGCGTGTG-3′ and 5′-GGGGTGTTGAAGGTCTCAAA-3′ for β-actin mRNA.
10
Quantification of Yeast Gene Expression
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Total RNA was isolated using a yeast total RNA extraction kit (Sangon Biotech Co. Shanghai, China). Isolated RNA was treated with RNase-free DNase I (Toyobo, Osaka, Japan) and cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix kit (Toyobo, Osaka, Japan) as described [13] . Real-time PCR was conducted on a Bio-Rad iCycler iQ (Bio-Rad, Hercules, CA, USA) using the THUNDERBIRD SYBR qPCR mix kit (Toyobo, Osaka, Japan). The primers for KmYME and the ACT1 internal control are shown in Additional le 2: Table . S1. The cycle threshold values (C T ) were determined and the relative fold differences were calculated using the 2 - ΔΔCT method [52] with ACT1 as the endogenous reference gene.
The fold change was shown as the sign of the log 2 transformed fold change (FC) values (log 2 FC).
The fold change was shown as the sign of the log 2 transformed fold change (FC) values (log 2 FC).
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