Rotor gene sybr green pcr master mix

Manufactured by Qiagen

Sourced in France, Germany, United States

The Rotor-Gene SYBR Green PCR master mix is a ready-to-use solution designed for real-time PCR amplification and detection of DNA sequences using the SYBR Green fluorescent dye. It contains all the necessary components, including DNA polymerase, dNTPs, and SYBR Green, for efficient and reliable real-time PCR reactions.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (9 mentions) Rotor gene q system, by Qiagen (8 mentions) Trizol, by Thermo Fisher Scientific (8 mentions) Rotor gene q, by Qiagen (5 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions) Tri reagent, by Euromedex (4 mentions) Rotor gene q 2plex, by Qiagen (4 mentions) Easy spin total rna extraction kit, by iNtRON Biotechnology (3 mentions) Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions) Maxime rt premix kit, by iNtRON Biotechnology (3 mentions) Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions) Quantitect primer assay, by Qiagen (2 mentions) Rotor gene sybr green pcr kit, by Qiagen (2 mentions) Improm 2 reverse transcription system, by Promega (2 mentions) Rotor gene q real time pcr cycler, by Qiagen (2 mentions) Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Agilent bioanalyzer, by Agilent Technologies (2 mentions) Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Improm iitm reverse transcription system, by Promega (2 mentions)

42 protocols using rotor gene sybr green pcr master mix

miRNA Expression and Pathway Regulation

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The expression profiling of 798 miRNAs was carried out by the nCounter human v3 miRNA expression assay (NanoString Technologies, Seattle, WA, USA), as previously described [24 (link)].
The expression levels of DICER1, DROSHA, TARBP2, DGCR8 and XPO5 were evaluated by RT-PCR using the QuantiTect Primer Assay (Qiagen Inc, Hilden, Germany), and following the manufacturer’s instructions. In detail, the retrotranscription step was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA), as previously reported [22 (link)]. Fifty ng of cDNA were then used in a reaction volume of 25 μL with the Rotor-Gene SYBR Green PCR Master Mix (Qiagen Inc, Hilden, Germany), and with specific primers. Amplification was performed in 40 cycles (denaturation at 95°C for 5 sec, annealing and elongation at 60°C for 10 sec). Actin beta (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as housekeeping genes, and thyroglobulin (TG) was used as specificity control. Each sample was amplified in double copy. The ΔΔC_t method was used to assess the relative quantification of the targets [25 ].

Poma A.M., Condello V., Denaro M., Torregrossa L., Elisei R., Vitti P, & Basolo F. (2019). DICER1 somatic mutations strongly impair miRNA processing even in benign thyroid lesions. Oncotarget, 10(19), 1785-1797.

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Analyzing microRNA Expression via qRT-PCR

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To validate the NGS data, the expression of microRNAs was analyzed via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) with the Rotor-Gene SYBR Green PCR Kit, miScript Primer Assays, and miScript Universal Primer, following reverse transcription of total RNA with the miScript II RT Kit (Qiagen; Hilden, Germany).
The qRT-PCR analysis was performed in technical replicates according to the manufacturer's instructions using the Rotor-Gene SYBR Green PCR Master Mix (Qiagen; Hilden, Germany). The packaged operating software was utilized for instrument control, data acquisition, and raw data analysis. The plates were run in relative quantification (ΔΔC_t) mode with triplicate measurements.
Amplification curves were analyzed using the packaged operating software, and assays were inspected for distinct melting curves. In addition, only assays detected with C_t < 35 were included in the data analysis. To calculate the relative expression levels of target microRNAs, the ΔΔC_t algorithm method was utilized. miR-423-3p and miR-423-5p were stably expressed across all samples, thus the average of their C_ts in each sample was used as the normalization factor. Assays were calibrated to the same normal skin sample.

Ning M.S., Kim A.S., Prasad N., Levy S.E., Zhang H, & Andl T. (2014). Characterization of the Merkel Cell Carcinoma miRNome. Journal of Skin Cancer, 2014, 289548.

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Quantifying Rice Transcriptome Changes

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Total rice RNA was isolated using the Easy-spin total RNA extraction kit (iNtRON Biotechnology) according to the manufacturer’s instructions. First-strand cDNAs were synthesized using 2 μg of total RNA and the ImProm-II Reverse Transcription System (Promega) with oligo (dT) primers. qRT-PCR reactions were performed using a Rotor-Gene Q real-time PCR cycler (Qiagen) and the gene-specific primers listed in Supplementary Table 11. Each PCR tube contained 5 μl of 2× Rotor-Gene SYBR Green PCR master mix (Qiagen), 25 ng of cDNA, and 15 pmol of each primer. The thermal cycling conditions were 10 min at 94 °C followed by 40 cycles of 15 s at 94 °C and 1 min at 60 °C.
The quality of extracted RNA samples was assessed using Bioanalyzer 2100 (Agilent). RNA-Seq libraries were prepared using the TruSeq RNA Library Prep Kit (Illumina) and sequenced using HiSeq2500 (Illumina). Raw sequence reads were trimmed to remove adaptor sequences, and those with a quality lower than 20 were removed using the NGS QC Toolkit v2.3.3^{49 (link)}. All reads were assembled and mapped via the use of HISAT2 v2.1.0^{50 (link)} and StringTie v1.3.5^{51 (link)}. DEGs in the MoHTR-OX lines were identified as the genes whose transcript abundance was ≥ 2-fold higher or ≤ 0.5-fold lower than their transcripts in Nakdong rice.

Kim S., Kim C.Y., Park S.Y., Kim K.T., Jeon J., Chung H., Choi G., Kwon S., Choi J., Jeon J., Jeon J.S., Khang C.H., Kang S, & Lee Y.H. (2020). Two nuclear effectors of the rice blast fungus modulate host immunity via transcriptional reprogramming. Nature Communications, 11, 5845.

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Quantitative Analysis of IGF2 CNV

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The CNV of IGF2 gene was analyzed using the qBiomarker CNV PCR Assay for Human chromosome 11 tile 10,752 (Qiagen). This assay is based on amplification and quantification of an IGF2 sequence in relation to two reference genes ZNF80 and GPR15 [40 (link)]. Quantitative PCR reactions were performed according to manufacturer’s protocol in triplicates in 20 μl reaction volume containing Rotor-Gene Sybr Green PCR Master Mix (Qiagen). Data generated by qPCR were analyzed using the commercially available qBaseplus software (Biogazelle NV, Zwijnaarde, Belgium). The reference genes ZNF80 and GPR15 were used for normalization of the relative CNV values. Three blood DNA samples from healthy donors, assumed to have normal diploid genomes, were used as additional calibrators for calculation of CNV in different PCR runs. A minimum 2-fold increase of IGF2 copy numbers in comparison to blood DNA samples was determined as a “gain”, and a decrease of IGF2 copy numbers (less than 0.5-fold) was determined as a “loss”. Values similar to those in blood DNA samples were considered as “normal”.

Schagdarsurengin U., Lammert A., Schunk N., Sheridan D., Gattenloehner S., Steger K., Wagenlehner F, & Dansranjavin T. (2017). Impairment of IGF2 gene expression in prostate cancer is triggered by epigenetic dysregulation of IGF2-DMR0 and its interaction with KLF4. Cell Communication and Signaling : CCS, 15, 40.

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Cardiac Cytokine Expression Analysis

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Gene expression of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) were measured in cardiac homogenates. RNA extraction was performed using TRIzol^® (Thermo Scientific) according to the manufacturer's instructions. Chloroform was added (0.2 mL/mL of TRIzol^®), and samples were mixed and centrifuged for 15 min at 12,000 g and 4°C. Aqueous phase containing RNA was collected, mixed with isopropanol to precipitate RNA, and centrifuged (12,000g, 4°C, 15 min). After centrifugation, the pellet was washed with ethanol 70% (v/v), dried, and suspended in water. RNA quantification and integrity were verified by measuring the ratio of optical density at 260 and 280 nm and by agarose gel migration, respectively. Two micrograms of total RNA was used for reverse transcription. The products of reverse transcription were used for reverse transcription quantitative polymerase chain reaction (RT‐qPCR) to evaluate gene expression. TaqMan low density array was used using 384‐well format plates on a 7900HT Fast Real Time PCR system (Applied Biosystems). Gene expression was performed using specific primers (sequences available on request) and Rotor‐Gene SYBR Green PCR master mix on a Rotor‐Gene Q System (Qiagen, Courtaboeuf, France). mRNA quantification was assayed using the ddCT method. β‐actin was used as the housekeeping gene.

Léger T., Charrier A., Moreau C., Hininger‐Favier I., Mourmoura E., Rigaudière J., Pitois E., Bouvier D., Sapin V., Pereira B., Azarnoush K, & Demaison L. (2017). Early sepsis does not stimulate reactive oxygen species production and does not reduce cardiac function despite an increased inflammation status. Physiological Reports, 5(13), e13231.

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Cardiac Gene Expression Analysis

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Total RNA were extracted from 50 mg of cardiac powder using TRIzol® (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions. RNA quantification and integrity were verified by measuring the ratio of optical density at 260 and 280 nm and by agarose gel, respectively. cDNA was synthesized from 2 μg of total RNA using a High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems (Thermo Scientific, Rockford, IL). The reverse transcription products were used for quantitative real-time polymerase chain reaction (qRT-PCR) using specific primers and Rotor-Gene SYBR Green PCR master mix on a Rotor-Gene Q system (Qiagen, Courtaboeuf, France). Messenger RNA (mRNA) was quantified using the standard curve of native cDNA and serial dilutions. mRNA expressions were determined for p53, PGC1-α, angiotensin-2, angiotensin-2 receptors-1a and -1b, pyruvate dehydrogenase 4, superoxide dismutase 2, glutathione peroxidase 4, catalase, ICAM-1, VCAM-1 and nitric oxide synthase 3. Primer sequences and PCR conditions can be made available on request (luc.demaison@inra.fr). β-actin and non-POU domain-containing octamer-binding protein (NoNo) genes were used as housekeeping genes.

Leger T., Hininger-Favier I., Capel F., Geloen A., Rigaudière J.P., Jouve C., Pitois E., Pineau G., Vaysse C., Chardigny J.M., Michalski M.C., Malpuech-Brugère C, & Demaison L. (2018). Dietary canolol protects the heart against the deleterious effects induced by the association of rapeseed oil, vitamin E and coenzyme Q10 in the context of a high-fat diet. Nutrition & Metabolism, 15, 15.

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Cytokine Gene Expression Analysis by Real-Time PCR

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To investigate the effects of WPC and WPC hydrolysates on cytokine gene expression, the mRNA
expression levels of IL-1β, IL-4, and IL-13 were analyzed using real-time PCR. To
perform real-time PCR, the extracted cRNA was mixed with a Rotor-Gene SYBR Green PCR Master
Mix (Qiagen, Germany), and -F and –R primers (10 pmol/µL) for each cytokine gene
of interest, and nuclease-free water. The primer gene sequences of the three cytokines
analyzed are presented in Table 1. A Rotor-Gene Q 2plx
(Qiagen, Germany) was used for real-time-PCR analysis, with the conditions for the
IL-1β reaction being as follows; 40 cycles of pre-denaturation at 95°C for 5
min, denaturation at 95°C for 5 min, annealing at 56°C for 30 sec, and
polymerization at 72°C for 60 sec, and 34 cycles of pre-denaturation of IL-4 and IL-13
at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 63°C for
45 sec, and polymerization at 72°C for 45 sec. The amplified products were examined
using the Delta Delta CT method, and each sample was calibrated to the expression level of the
housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each PCR product was
electrophoresed on a 1% agarose gel containing Red Safe (Intron Biotechnology Inc., Sungnam,
Korea) at 100 V for 20 min and photographs were taken under UV light using the UVD GelDoc-It
documentation system (UVP, Upland, CA, USA).

Kim H., Ahn S.I., Jhoo J.W, & Kim G.Y. (2018). Comparison of Allergic Parameters between Whey Protein Concentrate and Its Hydrolysate in Rat Basophilic Leukemia (RBL)-2H3 Cells. Korean Journal for Food Science of Animal Resources, 38(4), 780-793.

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Biofunctionalized Alginate Hydrogel Synthesis

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Sodium alginate, calcium chloride (CaCl2), silica solution (LUDOX® AM colloidal silica, 30 wt. % in H₂O), 2-(3,4-Dihydroxyphenyl)ethylamine hydrochloride (dopamine hydrochloride), 1-(3-Dimethylaminopropyl)-3-EthylcarbodiimideHydrochloride (EDC), and N-Hydroxy-Succinimide (NHS) werei all obtained from Sigma–Aldrich (St. Louis, MO). Cell culture media RPMI-1640 medium, fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Invitrogen Corporation (Carlsbad, CA). Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) was obtained from Millipore (Billerica, MA). Proteinase K, 95% ethyl alcohol, AW1 wash buffer, nuclease-free water, and SYBR Green Master Mix (2× Rotor Gene SYBR Green PCR Master Mix) were purchased from Qiagen Inc. (Valencia, CA). All other chemicals used in this study were obtained from Sigma-Aldrich and used without further purification unless otherwise noted.

Bu J., Lee T.H., Jeong W.J., Poellmann M.J., Mudd K., Eun H.S., Liu E.W., Hong S, & Hyun S.H. (2020). Enhanced detection of cell-free DNA (cfDNA) enables its use as a reliable biomarker for diagnosis and prognosis of gastric cancer. PLoS ONE, 15(12), e0242145.

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Quantitative detection of influenza A

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RNA was isolated from both AFs and CCSs using QIAmp Viral RNA mini kit (QIAGEN, Hilden, Germany) without using carrier RNA provided by the kit and reverse transcribed under the standard conditions of RevertAid Reverse Transcriptase (Thermo Scientific, Germany) using the Uni 12 primer [30 (link)]. Diluted cDNA (1:10) was used for the PCR reaction. A unique primer pair was designed to amplify the target sequence of the M gene of AIV type A generating a product of 150 bp (primer sequences are available on request). The qRT-PCR reactions were carried out according to the manufacturer’s instructions using the Rotor-Gene SYBR Green PCR master mix (QIAGEN, Hilden, Germany). A pJET 1.2 BVP/ H9N2-M plasmid was used as a positive control to develop a standard curve. The genome copy number was calculated based on the standard curve and expressed as log₁₀ genome copies/reaction.

Parvin R., Shehata A.A., Heenemann K., Gac M., Rueckner A., Halami M.Y, & Vahlenkamp T.W. (2015). Differential replication properties among H9N2 avian influenza viruses of Eurasian origin. Veterinary Research, 46(1), 75.

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RNA Extraction and qRT-PCR Analysis

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Total RNA were extracted from tissues using TRIzol® (Thermo Scientific) according to the manufacturer’s instructions. RNA quantification and integrity were verified by measuring the ratio of optical density at 260 nm and 280 nm and by agarose gel respectively. cDNAs were synthesized from 2 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit from Applied Biosystem (Thermo Scientific). The products of reverse transcription were used for Quantitative real time polymerase chain reaction (qRT-PCR) using specific primers and Rotor-Gene SYBR Green PCR master mix on a Rotor-Gene Q system (Qiagen, Courtaboeuf, France). Messenger RNA (mRNA) quantification was assayed using the standard curve of native cDNA and serial dilutions. Primer sequences and PCR conditions are available upon request (frederic.capel@inra.fr). Hypoxanthine guanine phosphoribosyltransferase (HPRT) and Non-POU-domain containing octamer binding protein (NoNo), gene were used as housekeeping gene in skeletal muscle and AT respectively.

Capel F., Geloen A., Vaysse C., Pineau G., Demaison L., Chardigny J.M., Michalski M.C, & Malpuech-Brugère C. (2018). Rapeseed oil fortified with micronutrients can reduce glucose intolerance during a high fat challenge in rats. Nutrition & Metabolism, 15, 22.

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