To examine the expression of inflammatory-related molecules by DCs and their effects on CD4+ T cells after exposure to Pg LPS, total RNA was extracted from DCs or CD4+ T cells at different time points after exposure to Pg LPS using RNAiso (Takara Bio Inc., Shiga, Japan). Complementary DNA (cDNA) was prepared via reverse transcription of the total RNA using a QuantiTect Reverse Transcription Kit (QIAGEN, Hilden, Germany). The diluted cDNA samples were analyzed via real-time reverse transcription polymerase chain reaction (RT-PCR). Real-time PCR was performed using the Rotor-Gene SYBR Green RT-PCR Kit (QIAGEN) and Corbett Rotor-Gene RG-3000A Real-Time PCR System (Biocompare, Sydney, Australia). We determined the copy numbers via the 2-Ct method using a calibrator. The sequences of the primer pairs are as follows: IL-6: 5′-TCAATTCCAGAAACCGCTATGA-3′ and 5′-CACCAGCATCAGTCCCA AGA-3′; Rorc: 5′-GCCTCCTGCCACCTTGAGT-3′ and 5′-TCTGCCTTCAGCTTTGCCTC-3′; Tbx21: 5′-CACTAAGCAAGGACGGCGAA-3′ and 5′-CCACCAAGACCACATCCACA-3′; Foxp3: 5′-GAAGCTGGGAGCTATGCAGG-3′ and 5′-TGGCTACGATGCAGCAAGAG-3′; GATA3: 5′-TTTACCCTCCGGCTTCATCCTCCT-3′ and 5′-TGCACCTGATACTTGAGGCACTCT-3′; IL-17: 5′-ACCTCAACCGTTCCACGTCA-3′ and 5′-CAGGGTCTTCATTGCGGTG-3′; Actin: 5′-AGAGGGAAATCGTGCGTGAC-3′ and 5′-CAATAGTGATGACCTGGCCGT-3 ′. For the data normalization, all gene expressions were normalized to actin.
Rotor gene sybr green rt pcr kit
Manufactured by Qiagen
Sourced in Germany, United States, Japan
The Rotor-Gene SYBR Green RT-PCR Kit is a real-time PCR reagent kit designed for quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis. It contains all the necessary components, including SYBR Green I dye, for the detection and quantification of RNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (7 mentions)
Quantitect reverse transcription kit, by Qiagen (6 mentions)
Quantitect primer assay, by Qiagen (4 mentions)
Rnaiso plus, by Takara Bio (3 mentions)
Lightcycler 480 2, by Roche (3 mentions)
Rotor gene q, by Qiagen (2 mentions)
Rotor gene rg 3000a real time pcr system, by Qiagen (2 mentions)
Direct zol rna miniprep kit, by Zymo Research (2 mentions)
Tri reagent, by Thermo Fisher Scientific (2 mentions)
Rnaiso, by Takara Bio (1 mentions)
Ptc 200 dna engine, by Bio-Rad (1 mentions)
Rneasy extraction kit, by Qiagen (1 mentions)
Rotor gene q pure detection version 2.1.0 build 9, by Qiagen (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
22 protocols using rotor gene sybr green rt pcr kit
1
Profiling Inflammatory Molecules in DCs and CD4+ T Cells
2
RNA Quantification via RT-qPCR
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Qiagen (Venlo, The Netherlands) RNase-free DNase I was used for DNase-treatment. We subjected 1.0 μg RNA to reverse transcription using TaqMan reverse transcription reagent (ABI). DNA Engine PTC-200 (MJ Research: Bio-Rad, Hercules, CA) was used for reverse transcription; RT conditions were as follows: 25 °C for 10 min, 1 °C for 30 min, and 95 °C for 5 min. Rotor-Gene Q (Qiagen) and a Rotor-Gene SYBR Green RT-PCR kit (Qiagen) were used for qPCR. qPCR conditions were as follows: (95 °C for 10 min, R °C for 15 s, 72 °C for 20 s), 40 cycles, R: annealing temperature. The melting-point curve was checked for nonspecific amplification and primer–dimer complexes. A relative standard was incorporated and target quantity was determined from a standard curve and divided by the endogenous quantity control.
3
Quantitative RT-PCR Analysis of Cardiac Gene Expression
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Total RNA was extracted from cardiac tissues with the RNeasy Extraction Kit (Qiagen®, USA). Quantitative RT-PCR assays were performed using Rotor-Gene SYBR Green RT-PCR Kit (Qiagen®, USA) on Rotor-Gene Q (Qiagen®, Valencia, CA, USA). Quantitative RT-PCR amplification conditions started with initial reverse transcription for the synthesis of cDNA for 10 minutes at 55°C and the resultant cDNA was then amplified by 40 cycles of PCR as follows: Denaturation at 95°C for 5 seconds, annealing at 55°C for 15 seconds and extension at 60°C for 15 seconds. GAPDH, the housekeeping gene, was used as a reference gene for normalization. Primers used for rat genes are presented in Table 1 . The values of the threshold cycle (Ct) were determined by Rotor-Gene Q-Pure Detection version 2.1.0 (build 9) (Qiagen®, USA). For each gene, the relative change in mRNA in samples was determined using the 2−ΔΔCt method (4) and normalized to the housekeeping gene GAPDH.
4
Real-Time PCR Analysis of Inflammatory Genes
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Total RNA was extracted using RNAiso Plus according to the manufacturer's instructions. 1 μg of total RNA was used for cDNA synthesis using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). After an initial amplification with a denaturation step at 95°C for 5 m, followed by 30–40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 10 s, and extension at 72°C for 30 s, a final extension at 72°C for 5 m was done upon completion of the cycling steps. The cDNA was amplified in duplicate using a Rotor-Gene SYBR Green RT-PCR Kit (Qiagen) with a Corbett Rotor-Gene RG-3000A Real-Time PCR System. The data were evaluated using the RG-3000A software program (version Rotor-Gene 6.1.93, Corbett, Sydney, Australia). The sequences of primer pairs were described as follows: IL-1β: 5′-CAACCAACAAGTGATATTCTCCATG-3′ and 5′-GATCCACACTCTCAGCTGCA-3′; inducible nitric oxide synthase (iNOS): 5′-GCCACCAACAATGGCAAC-3′ and 5′- CGTACCGGATGAGCTGTGAATT- 3′; TNF-α: 5′-ATGGCCTCCCTC TCAGTTC -3′ and 5′-TTGGTGGTTTGCTACGACGTG-3′; and IL-10: 5′-GACCAGCTGGACAACATACTGC TAA-3′ and 5′-GATAAGGATTGGCAACCCAAGTAA-3′. For data normalization, an endogenous control (actin) was assessed to control for the cDNA input, and the relative units were calculated by a comparative Ct method. All qRT-PCR experiments were repeated three times, and the results are presented as the means of the ratios ± SEM.
5
Quantitative Real-Time PCR Analysis
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Total RNA was isolated from the HMM cell lines using the RNeasy Plus Mini Kit protocol (Qiagen, Valencia, CA, USA). The quality and quantity of the total RNA were assessed using Nanodrop (Nanodrop Technologies, Wilmington, DE, USA). Total RNA of 500 ng was used to synthesize cDNA using QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA). Quantitative real-time PCR was performed using the Rotor-Gene SYBR Green RT-PCR Kit (Qiagen, Valencia, CA, USA). PCR conditions were as follows: 1 cycle at 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 s, and then 60 °C for 30 s. The expression level of each gene was normalized based on endogenous GAPDH expression. The analysis of relative gene expression was determined according to the 2−ΔΔCt method, as previously described [21 (link)]. The primer sequences used in this study are listed in Additional file 1 .
6
Hippocampal Gene Expression Analysis
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Rats were sacrificed, and brain tissue was isolated from the hippocampus on ice. Then, the hippocampi were frozen in liquid nitrogen and stored at −80°C. According to the manufacturer’s instructions, total RNA was extracted with RNAiso Plus (Takara, Japan). After the initial denaturation step at 95°C for 5 min, 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 10 s, and extension for 30 s were carried out. Rotor gene SYBR Green RT-PCR kit (Qiagen, Japan) with Corbett rotor gene RG-3000a real-time PCR system (Rotor-Gene 6.1.93, Corbett) was used. The data were evaluated using the RG-3000a software program (rotor gene version 6.1.93, Corbett). All RT-PCR experiments were repeated thrice. The primer sequences used were as follows: rat Brain-derived neurotrophic factor (BDNF): 5′-CCATAAGGACGCGGACTTGT-3′ and 5′-GAGGCTCCAAAGGCACTTGA-3′; rat Tyrosine Kinase receptor B (TrkB): 5′-ATTGACCCAGAGAACATCAC-3′ and 5′-CAGGAAATGGTCACAGACTT-3′; rat Protein kinase B-1 (AKT1): 5′-GTGGCAAGATGTGTATGAG-3′ and 5′-CTGGCTGAGTAGGAGAAC-3′; rat N-methyl-D-aspartate receptor 2B (NR2B): CGCATCTGTCCACCATT-3′ and 5′-GCATCAGGAAAGCCTCG-3′.
7
Quantifying PI3K, AMPK, and FOXO1 Expression
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The relative mRNA expression levels of the phosphatidylinositol 3-kinase (PI3K), adenosine monophosphate-activated protein kinase (AMPK), and forkhead box-O1 (FOXO1) genes were assessed using qRT-PCR. The RNA was obtained from the pancreas and liver utilizing Trizol Reagent (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol. Subsequently, the qRT-PCR was conducted employing the Rotor-Gene SYBR Green RT-PCR Kit (Qiagen, Montgomery, MD, USA), following the protocols supplied by the manufacturer. Briefly, 2x Rotor-Gene SYBR Green RT-PCR Master Mix, template RNA (≤100 ng/reaction), primers, and RNase-free water were mixed and put on ice. The cDNA synthesis was carried out at 55 °C for 10 min and then at 95 °C for 5 min for RNase deactivation. Afterward, the qRT-PCR was initiated, and the temperature and time for the annealing step were optimized for each reaction according to the primer used in the PCR reaction. The sequences of primers applied for the qRT-PCR are shown in Table 1 . The fold difference was computed using the Equation (2) −ΔΔct relative to β-actin, as reported earlier [44 (link),45 (link)].
8
Quantitative Real-Time PCR Protocol
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The total RNA 400 ng was used to synthesize cDNA using QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA). Quantitative real-time PCR was performed using the Rotor-Gene SYBR Green RT-PCR Kit (Qiagen). The PCR conditions were as follows: 95◦C for 5 min followed by 45 cycles of 95◦C for 10 seconds and ending at 60◦C for 30 seconds. Expression levels of each target gene were normalized to the endogenous GAPDH level. The relative gene expression was analyzed as described 24 (link). Primer sequences for the target genes selected for validation are documented in Table S4 .
9
RNA Extraction and qRT-PCR Analysis
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The total RNA was isolated using the RNeasy Mini kit (Qiagen, Manchester, UK) according to the manufacturer's instructions. First-strand synthesis and real-time qRT-PCR amplification (Roche LightCycler 480 II) were carried out in a one-step, single-tube format using the Rotor-Gene SYBR Green RT-PCR kit (Qiagen) and using validated primer pairs from Qiagen (Quantitect primer assay). The hydroxymethylbilane synthase (HMBS) gene was used as an internal standard. All reactions were run at least in technical triplicates, and mean ct values were transformed into virtual mRNA levels according to the formula: virtual mRNA level = 10 × ((ct(target) – ct(standard))/slope of the standard curve).
10
Placental Gene Expression Analysis by qPCR
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Gene expression of TGFβ1, VEGF-A, Angiotensinogen, Renin, ACE, AT1aR and AT2R were evaluated by quantitative real-time PCR (qPCR) using total RNA extracted from the maternal and fetal portions of the placenta followed by reverse transcription to obtain cDNA. The qPCR was performed using the Rotor-Gene SYBR® Green RT-PCR Kit (Catalog no. 204074, Qiagen). The following primers were used (Table 2 ):
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