Microamp optical

Manufactured by Thermo Fisher Scientific

Sourced in United States

The MicroAmp Optical is a high-quality microplate for use in real-time PCR and other fluorescence-based assays. It is designed to provide optimal optical performance and compatibility with a variety of real-time PCR instruments.

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23 protocols using microamp optical

Comprehensive Genomic Profiling via OncoScan

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Up to 12 ng/μl DNA was plated at 6.6 μl per well (maximum of 79.2 ng DNA per well) into MicroAmp Optical 96-well reaction plates (Life Technologies), which were either used immediately or frozen at −20 °C until needed.
The OncoScan assay utilises molecular inversion probe (MIP) technology,^{20 (link)} for the identification of CN alterations, loss of heterozygosity (LOH) and recurrent clinically actionable somatic mutations (SMs). MIP probes in this assay enable the capture of the alleles of over 220 000 SNPs distributed across the whole genome, with increased probe density within ~900 cancer genes. They also enable detection of 74 frequently tested somatic mutations in BRAF, KRAS, EGFR, IDH1, IDH2, PTEN, PIK3CA, NRAS and TP53. The assay was undertaken following the recommended OncoScan protocol as previously described.^{21 (link)}OSCHP files were generated by the OncoScan Console software (Affymetrix), using data from fluorescence intensity (CEL) files generated during scanning of OncoScan chips. OSCHP files were used as inputs for the SM Viewer Software v1.01.16304 (Affymetrix) for the detection of SMs in each sample and Nexus Express for OncoScan 3.0.1 (BioDiscovery, Hawthorne CA, USA) for the analysis of CN aberrations and LOH.

Togneri F.S., Ward D.G., Foster J.M., Devall A.J., Wojtowicz P., Alyas S., Vasques F.R., Oumie A., James N.D., Cheng K.K., Zeegers M.P., Deshmukh N., O'Sullivan B., Taniere P., Spink K.G., McMullan D.J., Griffiths M, & Bryan R.T. (2016). Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA. European Journal of Human Genetics, 24(8), 1167-1174.

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Quantification of Cell Adhesion and Matrix Metalloproteinase Genes

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Total RNA was extracted from AT-MSCs, BM-MSCs and HSC-3 cells using the NucleoSpin^® RNA isolation kit (Macherey-Nagel, Düren, Germany) and reverse-transcribed to cDNA using SuperScript™ IV VILO™ Master Mix (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania). All qRT-PCR reactions were conducted in triplicates on MicroAmp™ Optical 96-well reaction plates using TaqMan assays, with reactions carried out in QuantStudio 5 (all from Life Technologies, Carlsbad, CA, USA). The expression of the following genes was quantified: intercellular adhesion molecule 1 (ICAM1, assay ID Hs00164932_m1), integrin alpha-3 (ITGA3, assay ID Hs01076879_m1) and matrix metallopeptidase 1 (MMP1, assay ID Hs00899658_m1). Expression levels were normalized with the housekeeping gene, ribosomal protein lateral stalk subunit P0 (RPLP0, assay ID Hs99999902_m1), and calculated using the ddCt-method [40 (link)].

Sinha S., Narjus-Sterba M., Tuomainen K., Kaur S., Seppänen-Kaijansinkko R., Salo T., Mannerström B, & Al-Samadi A. (2020). Adipose-Derived Mesenchymal Stem Cells do not Affect the Invasion and Migration Potential of Oral Squamous Carcinoma Cells. International Journal of Molecular Sciences, 21(18), 6455.

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Quantitative Real-Time RT-PCR Analysis

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Total RNA, from either frozen liver tissues or cultured cells, was isolated by phenol-chloroform extraction. For each sample, 1 μg of total RNA was reverse-transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). cDNA was amplified in MicroAmp Optical 96-well reaction plates with SYBR Green PCR Master Mix (Applied Biosystems) on an Applied Biosystems Prism 7000 sequence detection system. Relative gene expression was calculated after normalization to a house-keeping gene (mouse or human 18S rRNA). All of the primers for real-time PCR were purchased from QIAGEN (Germantown, MD) with the following catalog numbers: PPH00340B for c-IAP; PPH00333B for c-FLIP; and PPH01716B for MnSOD.

Dou X., Li S., Hu L., Ding L., Ma Y., Ma W., Chai H, & Song Z. (2018). Glutathione disulfide sensitizes hepatocytes to TNFα-mediated cytotoxicity via IKK-β S-glutathionylation: a potential mechanism underlying non-alcoholic fatty liver disease. Experimental & Molecular Medicine, 50(4), 7.

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Gene Expression Analysis of Bacterial Cultures

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Bacteria
were cultured in TSB overnight. The overnight cultures were diluted
(1:100) in fresh TSB or TSB with 10% spent culture of S. epidermidis and grown for 8 h at 37 °C with
shaking. The pellets were collected by centrifuge at 4000g for 10 min and broken by FastPrep-24 (MP 116005500). After centrifugation,
the supernatant was used to isolate the total RNA and further synthesize
complementary DNA according to the manufacturer’s instructions
(Qiagen). The cDNA was used as a template for real-time PCR using
SYBR-green PCR reagents (Roche) with primers listed in Table S1. Reactions were performed in a MicroAmp
Optical 96-well reaction plate using a 7500 Sequence Detector (Applied
Biosystems). All qRT-PCR experiments were performed in triplicate
using gyrB as an internal control gene at the mRNA
level.

Nurxat N., Wang L., Wang Q., Li S., Jin C., Shi Y., Wulamu A., Zhao N., Wang Y., Wang H., Li M, & Liu Q. (2023). Commensal Staphylococcus epidermidis Defends against Staphylococcus aureus through SaeRS Two-Component System. ACS Omega, 8(20), 17712-17718.

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Quantitative Real-Time PCR Analysis of RNA Profiles

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Total RNA was extracted from frozen liver, epididymal fat, and muscle samples using TRIpure Isolation Reagent (Roche Diagnostics Belgium, Vilvoorde). cDNA was synthesized. Quantitative real-time PCR analysis was carried out as previously described [33 (link)]. Primer pairs for transcripts of interest F4/80, CD68, α2-HS glycoprotein (AHSG/fetuin-A), TNF-α, CD68, and RPL-19 chosen as an invariant standard were designed using the Primer Express design software (Applied Biosystems, Lennik, Belgium). Primer sequence for AHSG was 5′-TGGCCTGCAAGTTATTCCAAA-3′ (forward) and 5′-GCTGTGGGTACGGGACCTACT-3′ (reverse) [31 (link)]. The sequences of the other primers (F4/80, TNF-α, CD68, and RPL-19) have already been described [10 (link)]. All experimental tissues and standard curve samples were run in duplicate in a 96-well reaction plate (MicroAmp Optical, Applied Biosystems). Results are expressed as fold expression relative to expression in the control group using the ΔΔCt method.

Lanthier N., Lebrun V., Molendi-Coste O., van Rooijen N, & Leclercq I.A. (2022). Liver Fetuin-A at Initiation of Insulin Resistance. Metabolites, 12(11), 1023.

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Quantitative Gene Expression Analysis

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Total RNA, from either frozen liver tissue or cultured cells, was isolated with a phenol-chloroform extraction. For each sample, 1μg total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). The cDNA was amplified in MicroAmp Optical 96-well reaction plates with a SYBR Green PCR Master Mix (Applied Biosystems) on an Applied Biosystems Prism 7000 sequence detection system. Relative gene expression was calculated after normalization by a house-keeping gene (mouse or human 18S rRNA). The relevant primer sequences are shown in Table 1.

Ding L., Wo L., Du Z., Tang L., Song Z, & Dou X. (2017). Danshen protects against early-stage alcoholic liver disease in mice via inducing PPARα activation and subsequent 4-HNE degradation. PLoS ONE, 12(10), e0186357.

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Quantitative RT-PCR for S. aureus

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Total RNA of S. aureus or cells was extracted, and complementary DNA was synthesized from total RNA by using the QuantiTect reverse transcription system (Qiagen). Amplification of the resulting complementary DNA sample utilized the QuantiTect SYBR green PCR kit (Qiagen). The 7500 Sequence Detector (Applied Biosystems) was used to perform reactions in MicroAmp Optical 96-well reaction plates. Oligonucleotides used in this study are presented in Supplementary Table 2.

Wang Y., Zhao N., Jian Y., Liu Y., Zhao L., He L., Liu Q, & Li M. (2022). The pro-inflammatory effect of Staphylokinase contributes to community-associated Staphylococcus aureus pneumonia. Communications Biology, 5, 618.

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Quantitative RT-PCR Analysis of Hepatocytes

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Total RNA from hepatocytes was isolated with a phenol-chloroform extraction. For each sample, 1.0 μg total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). The cDNA was amplified in MicroAmp Optical 96-well reaction plates with a SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on an Applied Biosystems Prism 7000 sequence detection system. Relative gene expression was calculated after normalization by a house-keeping gene (mouse or human 18S rRNA).

Li J., Dou X., Li S., Zhang X., Zeng Y, & Song Z. (2015). Nicotinamide ameliorates palmitate-induced ER stress in hepatocytes via cAMP/PKA/CREB pathway-dependent Sirt1 upregulation. Biochimica et biophysica acta, 1853(11 Pt A), 2929-2936.

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Quantitative and Qualitative Gene Expression Analysis

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mRNA preparation was performed using Trizol reagent (Life Technologies), and cDNA was subsequently prepared using SuperScript II reverse transcriptase (Invitrogen, Life Technologies) according to the manufacturer’s protocol. One microliter of cDNA was mixed with 1× final concentration of Fast SYBR Green Master Mix (Applied Biosystems, Life Technologies) and 0.2 pmol/µL final concentration of respective forward and reverse primers in a total volume of 10 µL in an Applied Biosystems MicroAmp optical 96-well plate. The reaction was performed in an Applied Biosystems 7500 Fast real-time PCR machine. For semiquantitative RT–PCR, the reaction was performed in 0.2-mL PCR tubes (Biozym) using HT Master Taq polymerase, 0.2 mM dNTPs, MgCl-containing PCR buffer (Genaxxon Bioscience), and 1.5 µM forward and reverse primers.

Rosenbaum M., Andreani V., Kapoor T., Herp S., Flach H., Duchniewicz M, & Grosschedl R. (2014). MZB1 is a GRP94 cochaperone that enables proper immunoglobulin heavy chain biosynthesis upon ER stress. Genes & Development, 28(11), 1165-1178.

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qRT-PCR Analysis of Gene Expression

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Total RNA was isolated using an RNeasy Mini Kit (Qiagen) from cells grown to late logarithmic growth phase (4 h) or stationary growth phase (8 h) in tryptic soy broth (TSB). Complementary DNA was synthesized from total RNA using the QuantiTect reverse transcription system (Qiagen) according to the manufacturer’s instructions. The resulting complementary DNA and negative control samples were amplified using the QuantiTect SYBR green PCR kit (Qiagen). Reactions were performed in a MicroAmp Optical 96-well reaction plate using a 7500 Sequence Detector (Applied Biosystems). All qRT-PCR experiments were performed in triplicate, with gyrB as control. Oligonucleotides are listed in Table 2.

Li M., Dai Y., Zhu Y., Fu C.L., Tan V.Y., Wang Y., Wang X., Hong X., Liu Q., Li T., Qin J., Ma X., Fang J, & Otto M. (2016). Virulence determinants associated with the Asian community-associated methicillin-resistant Staphylococcus aureus lineage ST59. Scientific Reports, 6, 27899.

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