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Dna engine opticon 2 continuous fluorescence detection system

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The DNA Engine Opticon 2 Continuous Fluorescence Detection System is a laboratory instrument designed for real-time PCR applications. It features a continuous fluorescence detection system that allows for the monitoring of fluorescent signals during the amplification process.

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Lab products found in correlation

Platinum sybr green qpcr supermix udg, by Thermo Fisher Scientific (4 mentions) Iscript cdna synthesis kit, by Bio-Rad (4 mentions) Quantitect sybr green pcr kit, by Qiagen (4 mentions) Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions) M mlv rt, by Promega (3 mentions) Lightcycler 480 system, by Roche (2 mentions) Sybr green 1 master, by Roche (2 mentions) Takara ex taq rt pcr version 2.1 kit, by Takara Bio (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Dnase treatment and removal reagent, by Thermo Fisher Scientific (2 mentions) Rnasin ribonuclease inhibitor, by Promega (2 mentions) Computer software, by Bio-Rad (2 mentions) Sybr premix ex taq kit, by Takara Bio (1 mentions) Rnaiso reagent, by Takara Bio (1 mentions) Human genome u133 plus 2.0 array, by Thermo Fisher Scientific (1 mentions) M mlv reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions) Rneasy extraction kit, by Qiagen (1 mentions) Rt reagent kit, by Takara Bio (1 mentions) Cloned amv reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Trizol total rna isolation kit, by Sangon (1 mentions)

16 protocols using dna engine opticon 2 continuous fluorescence detection system

1

Quantitative Analysis of iASPP Expression

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Total RNA was extracted from the cell lines using the RNAiso reagent provided by Takara (Takara Bio, Inc., Otsu, Japan). cDNA was synthesized using myoblastosis virus reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) with 1 μg of total RNA. The qPCR assay was performed using DNA Engine Opticon 2 Continuous Fluorescence Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with the SYBR Premix ExTaq kit (Takara Bio, Inc.). Primers were designed using primer 5.0 version (Peimier Company, Canada) and synthesized by Sangon (Shanghai, China). Primers sequences used are as follow:
Primers iASPP was used as described by Wang et al. [8 (link)]. All experiments were carried out in triplicates.
Yu J., Li L, & Huang C. (2017). Downregulation of Inhibition of Apoptosis-Stimulating Protein of p53 (iASPP) Suppresses Cisplatin-Resistant Gastric Carcinoma In Vitro. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 23, 5542-5549.
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2

Transcriptome Analysis of Schwannoma Onset

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Thirty-seven samples from human tumor (schwannomas) tissues were obtained at the time of surgery in accordance with applicable human ethics regulations (USC IRB protocol # 97–157) and snap frozen in liquid nitrogen. Tumor specimens were divided into two groups (young and old) according to the patient's age of onset. The young-onset group included 21 specimens from patients whose age of onset was less than 16 years old (ranging from 7 to 16 years old). The old-onset group included 16 specimens from patients whose age of onset was 30 years and older (ranging from 30 to 67 years old). The average age of the young group was 12 years old. The average age of the old group was 43 years. RNA was extracted and cRNA were hybridized to Human Genome U133 Plus 2.0 Array (Affymetrix) for analysis of over 47,000 transcripts. The detailed protocol for the sample preparation and microarray processing is available from Affymetrix at http://www.affymetrix.com (Santa Clara, CA, USA). For real-time PCR, total RNA was extracted and RT-qPCR was performed in the DNA Engine Opticon 2 Continuous Fluorescence Detection System (Bio-Rad, Hercules, CA, USA).
Toren A., Reichardt J.K., Andalibi A., Hsu N.Y., Doherty J., Slattery W, & Mehrian-Shai R. (2014). Novel age-dependent targets in vestibular schwannomas. Human Genomics, 8(1), 10.
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3

Quantitative Analysis of Oocyte RNA

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Total RNA was extracted from GV, MI and MII oocytes (20 oocytes/sample) using the RNeasy extraction kit (Qiagen, Crawley, UK) as suggested by the manufacturer. Starting with 100 ng of RNA, complementary DNA (cDNA) was prepared using M–MLV reverse transcriptase kit (ThermoFisher Scientific, Waltham, MA, USA). Quantitative PCR analysis was performed using SYBR Green I Master and the LightCycler 480 System (Roche, Basel, Switzerland) on a DNA Engine Opticon 2 Continuous Fluorescence Detection System (BioRad, Hercules, CA, USA). The reaction was performed using the following qRT-PCR program: 95 °C for 10 min, followed by 40 amplification cycles of 95 °C for 10 s, 57 °C for 30 s, and 72 °C for 30 s. The primer used for the amplification of Cnr1, Cnr2, Gpr55, and Trpv1 [55 (link),56 (link)] were listed in Table 2 and all the data were normalized to the endogenous reference gene β-Actin. Relative quantitation of mRNAs was performed by the comparative ΔΔCt method [57 (link)].
Cecconi S., Rossi G., Oddi S., Di Nisio V, & Maccarrone M. (2019). Role of Major Endocannabinoid-Binding Receptors during Mouse Oocyte Maturation. International Journal of Molecular Sciences, 20(12), 2866.
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4

Quantitative RT-PCR Analysis of CYP1A Genes

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Total RNA was isolated using cold Trizol reagent and first-strand cDNA was synthesized using the RT reagent kit according to the manufacturer’s protocol (Takara, Shiga, Japan). Two microliters of cDNA were used for real time PCR using TaKaRa Ex Taq RT-PCR Version 2.1 kit (TaKaRa). Gene-specific PCR primers for CYP1A1/cyp1a1, CYP1A2/cyp1a2, and GAPDH/gapdh are listed in Table 2, and PCR signals were detected with a DNA Engine Opticon 2 Continuous Fluorescence Detection System (Bio-Rad, Hercules, CA, USA). PCR was monitored for 45 cycles using an annealing temperature of 60 °C. At the end of the PCR cycles, melt curve analysis and 2% agar electrophoresis was performed to assess the purity of the PCR products. Negative control reactions (no template) were routinely included to monitor potential contamination of reagents. Relative amounts of CYP1A1/cyp1a1 and CYP1A2/cyp1a2 mRNA were normalized to that of GAPDH/gapdh mRNA.
Wang K., Feng C., Li C., Yao J., Xie X., Gong L., Luan Y., Xing G., Zhu X., Qi X, & Ren J. (2015). Baicalin Protects Mice from Aristolochic Acid I-Induced Kidney Injury by Induction of CYP1A through the Aromatic Hydrocarbon Receptor. International Journal of Molecular Sciences, 16(7), 16454-16468.
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5

Quantification of hSETD1A mRNA Expression

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Total RNA was isolated by using a UNIQ-10 column and TRIzol Total RNA Isolation Kit (Sangon, Shanghai, China). One microgram of total RNA was used for reverse transcription in a reaction volume of 20 μl using Cloned AMV Reverse Transcriptase (Invitrogen). Two microliters of cDNA was used for real-time PCR using TaKaRa Ex Taq RT-PCR Version 2.1 kit (TaKaRa, Shiga, Japan). Gene-specific PCR primers for hSETD1A and GAPDH are listed in Table 2, and PCR signals were detected with a DNA Engine Opticon 2 Continuous Fluorescence Detection System (Bio-Rad, Hercules, CA, USA). PCR was monitored for 45 cycles using an annealing temperature of 60°C. At the end of the PCR cycles, melt curve analysis and 2% agar electrophoresis were performed to assess the purity of the PCR products. Negative control reactions (no template) were routinely included to monitor potential contamination of reagents. Relative amounts of hSETD1A mRNA were normalized to that of GAPDH mRNA.
Cheng X.S., Sun S.B., Zhong F., He K, & Zhou J. (2016). Knockdown of Histone Methyltransferase hSETD1A Inhibits Progression, Migration, and Invasion in Human Hepatocellular Carcinoma. Oncology Research, 24(4), 239-245.
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6

Quantitative PCR Analysis of Sertoli Cell mRNA

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Messenger RNAs were extracted from control or BPA-exposed Sertoli cells using TRIzol Plus RNA Purification Kit (Invitrogen, Carlsbad, CA, USA), as per manufacturer’s instructions, and were quantitated spectrophotometrically (NanoDrop, Thermo Scientific ND-2000c). One μg of total mRNA was reverse-transcribed to cDNA, using SuperScript Vilo™ Master Mix (Invitrogen, Carlsbad, CA, USA). Quantitative PCR analysis was performed using SYBR Green I Master and the LightCycler 480 System (Roche, Basel, Switzerland) on a DNA Engine Opticon 2 Continuous Fluorescence Detection System (BioRad, Hercules, CA, USA). The reaction was performed using the following qRT-PCR program: 95 °C for 10 min, followed by 40 amplification cycles of 95 °C for 10 s, optimal annealing temperature (see Table 1) for 30 s, and 72 °C for 30 s. The primers used for the amplification are listed in Table 1, with pertinent references. All data were normalized to the endogenous reference gene β-actin. Relative quantitation of mRNAs was performed by the comparative ΔΔCt method [22 (link),86 (link)].
Rossi G., Dufrusine B., Lizzi A.R., Luzi C., Piccoli A., Fezza F., Iorio R., D’Andrea G., Dainese E., Cecconi S, & Maccarrone M. (2020). Bisphenol A Deranges the Endocannabinoid System of Primary Sertoli Cells with an Impact on Inhibin B Production. International Journal of Molecular Sciences, 21(23), 8986.
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7

Total RNA Extraction and qPCR Analysis

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Total RNA was extracted using RNeasy Lipid or Mini Kits (Qiagen Inc., Valencia, CA, USA) for tissue and cell culture samples, respectively, following the supplier’s instructions. RNA content was quantified using NanoDrop technology (Fisher Scientific SAS, Illkirch Cedex, France) and quality checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA samples were treated with DNase Treatment and Removal Reagents (Thermo Fisher, Waltham, MA, USA) and reverse-transcribed for 1 h at 37 °C with 150-ng random primers, 0.5-mM dNTPs, 20 units of RNAsin Ribonuclease Inhibitor and 200 units of M-MLV RT (Promega, Madison, WI, USA). Primer sequences and amplification conditions are reported in Table 2. Real-time PCR was carried out with Platinum® SYBR® Green qPCR SuperMix-UDG (Thermo Fisher) on a DNA Engine OpticonTM 2 Continuous Fluorescence Detection System (MJ Research, Waltham, MA, USA). All experiments were performed in duplicate. Samples (5 ng of cDNA) were normalized by 18S rRNA content and reported as an arbitrary unit (a.u.) ratio or as a fold increase/decrease with respect to the control.
Sanna M., Borgo C., Compagnin C., Favaretto F., Vindigni V., Trento M., Bettini S., Comin A., Belligoli A., Rugge M., Bassetto F., Donella-Deana A., Vettor R., Busetto L, & Milan G. (2020). White Adipose Tissue Expansion in Multiple Symmetric Lipomatosis Is Associated with Upregulation of CK2, AKT and ERK1/2. International Journal of Molecular Sciences, 21(21), 7933.
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8

Quantitative PCR Assay for Gene Expression

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PCR was carried out with Platinum SYBR Green qPCR SuperMix-UDG (Thermo Fisher Scientific) on DNA Engine OpticonTM2 Continuous Fluorescence Detection System (MJ Research, MA, USA). Reaction conditions and primer sequences were reported in Table 4. Each sample (5 ng of cDNA) was assayed in duplicate and quantified using a standard curve method. Results were normalized to HMBS mRNA content and reported as arbitrary unit ratio (target/housekeeping). The melting curve analysis and a positive control (UCP1: VAT surrounding pheochromocytoma, PPARGC1A and TFAM: liver, TBX1: muscle, SLC27A1: white adipose tissue) were always included to check PCR specificity.
Bettini S., Favaretto F., Compagnin C., Belligoli A., Sanna M., Fabris R., Serra R., Dal Prà C., Prevedello L., Foletto M., Vettor R., Milan G, & Busetto L. (2019). Resting Energy Expenditure, Insulin Resistance and UCP1 Expression in Human Subcutaneous and Visceral Adipose Tissue of Patients With Obesity. Frontiers in Endocrinology, 10, 548.
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9

Quantifying Gene Expression in Cartilage

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Total RNA was isolated from the tibial cartilages of 6- and 12-month old animals with the RNeasy mini kit (QIAGEN, Valencia, CA, USA) as previously described by Wei et al21 (link). 1 μg total of RNA was used for each reverse transcriptase with the iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Real-time quantitative PCR amplification was performed using QuantiTect SYBR Green PCR kit (QIAGEN, Valencia, CA, USA) with the DNA Engine Opticon 2 Continuous Fluorescence Detection System (MJ Research, Waltham, MA, USA). Primers used in amplification of target genes’ mRNA are: 3’-TTCTACCTGGCCTTCTGC-5’ and 3’-GCAGTACATCTCCAGCCT-5’. IGF-1 mRNA level was normalized to housekeeping gene 18S RNA levels. Since the level of 18S RNA is constant in all the cells, the normalized values reflected the relative level of the target genes’ mRNA in each cell regardless of the cell number. The 18S RNA was amplified at the same time and used as an internal control. The cycle threshold (Ct) values for 18S RNA and that of samples were measured and calculated by computer software (MJ Research, Waltham, MA, USA). Relative transcription levels were calculated as x = −2ΔΔCt, in which Ct = E - C, and E = Ctexp-Ct18s; C = Ctctl-Ct18s.
WEI F.Y., LEE J.K., WEI L., QU F, & ZHANG J.Z. (2017). Correlation of insulin-like growth factor 1 and osteoarthritic cartilage degradation: a spontaneous osteoarthritis in guinea-pig. European review for medical and pharmacological sciences, 21(20), 4493-4500.
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10

RNA Extraction and qRT-PCR Analysis

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Total RNA was extracted using RNeasy Lipid or Mini Kits (QIAGEN) following the supplier's instructions. For each sample, 1 µg of RNA was treated with DNase Treatment & Removal Reagents (Ambion, Inc, Austin, TX, USA) and reverse-transcribed for 1 h at 37°C with 150 ng random hexamers, 0.5 mM dNTPs, 20 units of RNAs in Ribonuclease Inhibitor and 200 units of M-MLV RT (Promega, Madison, WI, USA). Oligonucleotide sequences and amplification conditions are reported in the Table S1. Real Time PCR was carried out with Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) on a DNA Engine Opticon 2 Continuous Fluorescence Detection System (MJ Research, MA, USA). Duplicate 5 ng cDNA samples were normalized by Rn18s (18S rRNA) content and reported as arbitrary units ratio.
Favaretto F., Milan G., Collin G.B., Marshall J.D., Stasi F., Maffei P., Vettor R, & Naggert J.K. (2014). GLUT4 Defects in Adipose Tissue Are Early Signs of Metabolic Alterations in Alms1GT/GT, a Mouse Model for Obesity and Insulin Resistance. PLoS ONE, 9(10), e109540.
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