Opticon 2 real time pcr detector

Manufactured by Bio-Rad

Sourced in United States

The Opticon 2 Real-Time PCR detector is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The device is capable of detecting and measuring the amplification of target DNA sequences in real-time during the PCR process.

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Lab products found in correlation

Rneasy kit, by Qiagen (2 mentions) Realmastermix, by Tiangen Biotech (2 mentions) 96 well thin wall pcr plate, by Bio-Rad (1 mentions) Sypro orange, by Thermo Fisher Scientific (1 mentions) Sypro orange, by Bio-Rad (1 mentions) Iq sybr green supermix reagent, by Bio-Rad (1 mentions) Bulge loop mirna qpcr primer set, by RiboBio (1 mentions) Rna extraction kit, by Transgene (1 mentions) Reverse transcription kit, by Transgene (1 mentions) Fluorescence quantitative pcr kit, by Transgene (1 mentions)

7 protocols using opticon 2 real time pcr detector

Gene Expression Analysis of Inflammatory Markers

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Total RNA from the heart and spleen were isolated using the RNeasy kit (Qiagen Inc, Valencia, CA, USA). mRNA expression of IL-1β, MCP-1, TNF-α, T-bet, IFN-γ, GATA3, IL-4, RORγt, IL-17A, TGF-β1, IL-6, Foxp3, IL-10 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected by real-time polymerase chain reaction (PCR) with the Opticon 2 Real-Time PCR detector (Bio-Rad) using the primers as previously described [19 (link), 42 (link)-45 (link)] and below: GATA3: forward 5′-TCTGGAGGAGGAACGCTAAT-3′, reverse 5′-TTCGGGTCTGGATGCCTTCTTT-3′; IL-4: forward 5′-TTCTCGAATGTACCAGGAGCCA-3′, reverse 5′-TCGTTGCTGTGAGGACGTTT-3′; IL-10: 5′-AAGGGTTACTTGGGTTGCCA-3′, reverse 5′-TGCTCCACTGCCTTGCTCTTAT-3′; RORγt: forward 5′-TGTCCTGGGCTACCCTACTG-3′, reverse 5′-GTGCAGGAGTAGGCCACATT-3′. The ratio of interested mRNA against GAPDH was calculated and expressed as the mean ± standard error of the mean (SEM).

Wang Y.Y., Jiang H., Wang Y.C., Huang X.R., Pan J., Yang C., Shou Z.F., Xiang S.L., Chen D.J., Lan H.Y, & Chen J.H. (2015). Deletion of Smad3 improves cardiac allograft rejection in mice. Oncotarget, 6(19), 17016-17030.

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Thermal Shift Assay of DUBm Mutants

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Thermal shift assay curves were collected using an Opticon 2 real-time PCR detector (BioRad) to measure the fluorescence of SYPRO Orange (Molecular Probes) in the presence of wild type DUBm (WT), DUBm-Sgf73^Y57A, DUBm-Sgf73^N59D and DUBm-Sgf73^E79A. Proteins were centrifuged for 30 minutes at 25000g at 4°C before sample preparation. DUBm complex (final concentration at 0.5 mg/ml) and SYPRO Orange dye (diluted 2000 folds to final concentration 2.5X) were mixed in the same buffer as K48-linked cleavage assay and added to 96-well thin-wall PCR plate (Bio-Rad). The temperature was increased from 5°C to 95°C in increments of 1°C and the fluorescence signals were collected. The wavelengths for excitation and emission were 490 and 575 nm, respectively.

Yan M, & Wolberger C. (2014). Uncovering the role of Sgf73 in maintaining SAGA Deubiquitinating Module Structure and Activity. Journal of molecular biology, 427(8), 1765-1778.

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ChIP Assay of Groucho Binding

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Eye discs of 3rd instar larvae were dissected in ice-cold PBS. Samples were double fixed with 1.5 mM DSP for 40 minutes and 1.8% formaldehyde for 15 minutes (Martinez and Arnosti, 2008 (link)). After double fixation, following steps are the same as the protocol described previously (Austin et al., 1999 (link)), except that SDS was omitted from the solutions. Sixty pairs of eye discs and 20 ul Gro antibody (concentrated, from Hybridoma Bank) were used for each ChIP assay. Real time RT-PCR (qPCR) was determined using the Thermo Scientific^™ DyNAmo^™ SYBR^™ Green qPCR Kit on an Opticon 2 Real-time PCR detector (BioRad). qPCR with each pair of primers was done in triplicate. The efficiency of Gro binding was shown by the ratio of ChIP DNA and input DNA content determined by qPCR. Primers include: rho-Gchip-1-F, 5′-AAGAACGCGAGTCAGCATTT-3′; rho-Gchip-1-R, 5′-CGTCCTCTGTCCTCCATTTC-3′; rho-Gchip-2-F, 5′-CTGGTCATGGGCATGTAGTG-3′; rho-Gchip-2-R, 5′-TTCAGAGTCCTCGTCCTCGT-3′; rho-Gchip-3-F, 5′-CAGTGAATAACCAGCGACGA-3′; rho-Gchip-3-R, 5′-GCACATCCGGATCTTGTCTT-3′; rho-Gchip-NC-F, 5′-TTCAACGGTGCATGAATGAT-3′; rho-Gchip-NC-R, 5′-ACTAGTCCGAGCGATTGCAG-3′; ato5′-1-F, 5′-TGTGCCCAAAGGAATAATCA-3′; ato5′-1-R, 5′-TCAAGGGTTGGACAAACAAA-3′; ato5′-2-F, 5′-GAGGGCTAAGGTGAAGGTCA-3′; ato5′-2-R, 5′-CAATTGATACGCTTGTTGCC-3′.

Zhang T, & Du W. (2015). Groucho restricts rhomboid expression and couples EGFR activation with R8 selection during Drosophila photoreceptor differentiation. Developmental biology, 407(2), 246-255.

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Real-time PCR Analysis of Gene Expression

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The total RNA was extracted from the HRGECs using the RNeasy kit according to the manufacturer’s instructions (Qiagen Inc., Valencia, CA, USA), and the cDNA was synthesized. A real-time PCR was performed with the Opticon®2 Real-Time PCR detector (Bio-Rad, Hercules, CA, USA) using the IQ SYBR green supermix reagent (Bio-Rad) as described previously
[17 (link)]. The primers used to amplify the mRNAs of TNF-α, TGF-β1, and GAPDH are listed in Table
2. The housekeeping gene GAPDH was used as an internal standard. The ratios of specific mRNA:GAPDH mRNA were calculated and expressed as the mean ± SEM.

Huang Y., Liu Y., Li L., Su B., Yang L., Fan W., Yin Q., Chen L., Cui T., Zhang J., Lu Y., Cheng J., Fu P, & Liu F. (2014). Involvement of inflammation-related miR-155 and miR-146a in diabetic nephropathy: implications for glomerular endothelial injury. BMC Nephrology, 15, 142.

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Quantitative Analysis of p53 and miRNA

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Quantitative real-time PCR (qRT-PCR) was processed by Real Master Mix (Tiangen Biotech, Co. Ltd) using a Bio-Rad Opticon2 Real-Time PCR Detector (Bio-Rad Laboratories). The primers are shown below.

Forward primer for p53 RT-PCR: 5′-TCAACAAGATGTTTTGCCAACTG-3′

Reverse primer for p53 RT-PCR: 5′-ATGTGCTGTGACTGCTTGTAGATG-3′

Forward primer for GAPDH RT-PCR: 5′-TGCACCACCAACTGCTTAGC-3′

Reverse primer for GAPDH RT-PCR: 5′-GGCATGGACTGTGGTCATGAG-3′

For miRNA detection, primers and protocols were supplied with the Bulge-Loop miRNA qPCR Primer Set (Ribobio). According to the manufacturer's instruction, the kit we used is based on the 2^−△△Ct method to test the relative amount of miR-19a and miR-19b. After total RNA of cells of the experimental group was extracted, miR-19a, miR-19b, and U6 were measured by different groups of primers, respectively, by qRT-PCR. Then the amount of miR-19a and miR-19b relative to U6 was calculated, respectively. Finally, these ratios were further normalized by the corresponding miRNA ratios in the control group. We repeated the experiment three times.

Fan Y., Yin S., Hao Y., Yang J., Zhang H., Sun C., Ma M., Chang Q, & Xi J.J. (2014). miR-19b promotes tumor growth and metastasis via targeting TP53. RNA, 20(6), 765-772.

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Quantitative Real-Time PCR Analysis

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Quantitative real-time PCR (qRT-PCR) was processed by Real Master Mix (Tiangen Biotech, Co. Ltd) using Bio-Rad Opticon2 Real-Time PCR Detector (Bio-Rad Laboratories). The primers are shown in Table 1.

Hao Y., Yang J., Yin S., Zhang H., Fan Y., Sun C., Gu J, & Xi J.J. (2014). The synergistic regulation of VEGF-mediated angiogenesis through miR-190 and target genes. RNA, 20(8), 1328-1336.

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VEGF Expression Analysis in Colon Cells

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HCT116 colon cells were incubated with PBS, Lipo3000 transfection of miR-190-Cy7, PPDCNPs (only miR-NC), PPDCNPs (only miR-190-Cy7), and m-PPDCNPs (only miR-190-Cy7) for 24 h or 48 h. The total RNA of each group was extracted by an RNA extraction kit (Transgene, catalog no. ER501), and cDNA was synthesized by a reverse transcription kit (Transgene, catalog no. AH341). Primer sequences were designed and amplified using a fluorescence quantitative PCR kit (Transgene, catalog no. AQ141), and the expression level of VEGF and GADPH were detected by the Bio-Rad Opticon2 Real-Time PCR Detector (Bio-Rad).
Human VEGF RT Primers: F 5′-GCG TGC TAA TGG TGG AAA C; R 5′-CGG TGA CAT CAA AAG ATA ACC AC.
Human GAPDH RT Primers: F 5′-TGT GGG CAT CAA TGG ATT TGG; R 5′-ACA CCA TGT ATT CCG GGT CAA T.

Zhu S., Li Z., Zheng D., Yu Y., Xiang J., Ma X., Xu D., Qiu J., Yang Z., Wang Z., Li J., Sun H., Chen W., Meng X., Lu Y, & Ren Q. (2022). A cancer cell membrane coated, doxorubicin and microRNA co-encapsulated nanoplatform for colorectal cancer theranostics. Molecular Therapy Oncolytics, 28, 182-196.

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