Microamp fast optical 96 well reaction plate

Manufactured by Thermo Fisher Scientific

Sourced in United States, Italy, Belgium, Canada

The MicroAmp Fast Optical 96-Well Reaction Plate is a laboratory equipment designed for use in real-time PCR applications. It is a 96-well microplate with an optical design that enables efficient light transmission and detection during the PCR process.

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124 protocols using microamp fast optical 96 well reaction plate

Exosomal microRNA Profiling by TaqMan

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Exo-miRNAs were analyzed by the TaqMan Array Card Technology. Briefly, 50 ng of RNA was reverse transcribed with the TaqMan® microRNA Reverse Transcription Kit, using the Megaplex^TM RT primers Human Pool A (Thermo Fisher Scientific, Monza, MB, Italy). Pre-amplification of cDNA was performed with TaqMan® PreAmp Master Mix and Megaplex^TM Pre-Amp primers Human Pool A. The pre-amplification product was diluted according to the manufacturer’s instructions and used to perform microRNA profiling on the ViiA^TM 7 Real-Time PCR System. Briefly, 9 μL of the diluted pre-amplified product was mixed with 450 μL TaqMan® Universal Master Mix II, No UNG (Thermo Fisher Scientific, Monza, MB, Italy), and 441 μL of nuclease-free water. 100 μL of the PCR reaction mix was dispensed into each well of the TaqMan® Array human microRNA A card (Thermo Fisher Scientific, Monza, MB, Italy), enabling the quantification of 381 human miRNAs. The validation was performed with individual qPCR assays based on specific TaqMan miRNAs Assays (Thermo Fisher Scientific, Monza, MB, Italy). Samples were run in triplicate on MicroAmp Fast Optical 96-well reaction plate (Thermo Fisher Scientific, Monza, MB, Italy).

Morini M., Cangelosi D., Segalerba D., Marimpietri D., Raggi F., Castellano A., Fruci D., Font de Mora J., Cañete A., Yáñez Y., Viprey V., Corrias M.V., Carlini B., Pezzolo A., Schleiermacher G., Mazzocco K., Ladenstein R., Sementa A.R., Conte M., Garaventa A., Burchill S., Luksch R., Bosco M.C., Eva A, & Varesio L. (2019). Exosomal microRNAs from Longitudinal Liquid Biopsies for the Prediction of Response to Induction Chemotherapy in High-Risk Neuroblastoma Patients: A Proof of Concept SIOPEN Study ‖. Cancers, 11(10), 1476.

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Thermal Shift Assay for Protein Stability

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Purified proteins at 0.5 mg/ml were mixed with SYPRO Protein Gel Stains (Thermo Fisher Scientific) at 1:500 dilution. 20 µl of the protein/SYPRO mixture were aliquoted to MicroAmp Fast Optical 96-Well Reaction Plate (Thermo Fisher Scientific). The experiments with temperature ramp from 25 to 95°C and 0.5°C/step were measured with The Applied Biosystems StepOnePlus 96-well qPCR system (Thermo Fisher Scientific). The melt curve data were analyzed with the StepOne Software (Thermo Fisher Scientific) and the melting temperatures of each construct were determined by taking the lowest point of the negative derivative of normalized fluorescence. Three measurements from each mutant were normalized, averaged, and plotted with Excel.

Lin D.Y., Kueffer L.E., Juneja P., Wales T.E., Engen J.R, & Andreotti A.H. (2024). Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states. eLife, 12, RP89489.

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RT-qPCR Gene Expression Analysis

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RNA extraction was performed using the RNeasy plant mini kit (Qiagen Ltd., Manchester, United Kingdom) in accordance with manufacturer’s instructions. RNA was stored at −80°C if not used immediately for RT-qPCR. TaqMan® RNA-to-CT ™ 1-Step Kit (Thermo Fisher Scientific, Basingstoke, United Kingdom) was used for gene expression analysis according to manufacturer’s instruction. Briefly, kit components and the primers pair (p21/CDKN1A:#Hs01121172_m1,GAPDH:#Hs99999905_m1, Thermo Fisher Scientific) were combined in a MicroAmp™ Fast Optical 96-Well Reaction Plate, 0.1 mL (10μLTaqMan® RT-PCR Mix (2x), 1 μL TaqMan® gene expression assay (20X), 0.5 μL TaqMan® RT Enzyme Mix (40x), 5 μL RNA template [up to 1 μg), 3.5 μL RNase-free H2O (up to Σ20μL)] was covered with MicroAmp optical adhesive film, briefly centrifuged. The PCR reaction was carried out using the Applied Biosystems StepOnePlus™ Real-Time PCR System with the run cycle (x1 reverse transcription, 30 min at 48°C, x1 cycle enzyme activation, 10 min at 95°C, x40 cycles (Denaturation 15 s at 95°C and Data collection, 1 min at 60°C). Fold change in gene expression vs. GAPDH was calculated as ΔCT.

Ito K., Daly L, & Coates M. (2023). An impact of age on respiratory syncytial virus infection in air-liquid-interface culture bronchial epithelium. Frontiers in Medicine, 10, 1144050.

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Detection of Borrelia burgdorferi in Tissues

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At necropsy, the bladder, left ear, left tibiotarsal joint, inferior half of the heart, and left popliteal and medial iliac lymph nodes were collected and frozen in liquid nitrogen. DNA was extracted from tissues using a DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions. DNA samples were diluted to match the lowest concentration sample and 5 µL of DNA was added to each well of a 96-well MicroAmp™ Fast Optical 96-Well Reaction Plate (ThermoFisher) containing 10 µL TaqMan Fast Advanced MasterMix and 1 µL of either Borrelia burgdorferi FlaB custom gene assay (Forward primer: 5’-TCTTTTCTCTGGTGAGGGAGCT-3’, Reverse primer: 5’-TCCTTCCTGTTGAACACCCTCT-3’, ThermoFisher) or mouse β-actin gene assay (ID: Mm02619580_g1, ThermoFisher). Reaction volumes were topped to 20 µL with nuclease-free water. qPCR reactions were performed using an Applied Biosystems™ 7500 Fast Real-time PCR instrument. Standard curves of plasmids encoding mouse β-actin or extracted B. burgdorferi genomic DNA were used to determine the number of FlaB copies per 1 × 10⁶ β-actin copies.

Pfeifle A., Thulasi Raman S.N., Lansdell C., Zhang W., Tamming L., Cecillon J., Laryea E., Patel D., Wu J., Gravel C., Frahm G., Gao J., Chen W., Chaconas G., Sauve S., Rosu-Myles M., Wang L., Johnston M.J, & Li X. (2023). DNA lipid nanoparticle vaccine targeting outer surface protein C affords protection against homologous Borrelia burgdorferi needle challenge in mice. Frontiers in Immunology, 14, 1020134.

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Quantification of Enterotoxin Genes by qPCR

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Primers used for uspA, STa, STb and LT toxins quantification are listed in Table 2. qPCR was performed on a 7500 Fast real-time PCR system (Thermo Fisher Scientific) using MicroAmp Fast Optical 96-well reaction plate (Thermo Fisher Scientific). qPCR reactions contained 10 μL QuantiFast SYBR green master mix (Thermo Fisher Scientific), 2 μL (10 μM) primers, 1 μL template DNA, and water to a final volume of 20 μL. PCR conditions were as follows: initial denaturation 5 min at 95 °C, 40 cycles of denaturation at 95 °C for 30s, annealing at corresponding temperature (Table 2) for 30s and followed by 30 extension at 72 °C. At the melting stage, temperature increased with a speed of 0.5 °C/10s from 55 to 95 °C. To calibrate qPCR assays, target genes were amplified from chromosomal DNA of E. coli ECL13998, purified by agarose electrophoresis, and the concentration was determined by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). The calibration of the qPCR assays was performed on the same instrument platform, with the same reagents and on the same day as the respective qPCR or HRM-qPCR assays.

Wang W., Zijlstra R.T, & Gänzle M.G. (2017). Identification and quantification of virulence factors of enterotoxigenic Escherichia coli by high-resolution melting curve quantitative PCR. BMC Microbiology, 17, 114.

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DENV2 NS3 Protein Thermal Shift Assay

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The protein thermal-shift assay (PTSA) was conducted using an Applied Biosystem 7500 Fast Real-Time PCR System (ThermoFisher Scientific) from 25 to 80 °C. The DENV2 His-MBP-NS3 or each mutant (final concentration of 2.5 μM in 1x PBS) was mixed with erythrosin B to attain a 4.8 μM final concentration in 1.6% DMSO in the MicroAmp^® Fast Optical 96-Well Reaction Plate (ThermoFisher Scientific). Thermal denaturation was monitored using SYPRO Orange (Life Technologies) according to manufacturer’s manual. The denaturation of the proteins was monitored by following the increase of the fluorescence emitted by the probe that binds exposed hydrophobic regions of the denatured protein. The melting temperature (T_m) was calculated as the mid-log of the transition phase from the native to the denatured protein, using a derivative model in the Protein Thermal Shift^™ Software v1.0 (ThermoFisher Scientific). The reference unfolding temperature of proteins in 1.6% DMSO (T_m-DMSO) was subtracted from the values in the presence of each compounds (T_m-comp) to obtain thermal shifts, ΔTm = T_m-comp – T_m-DMSO. Compounds were considered to be binders when ΔT_m > 0.5 °C.

Li Z., Sakamuru S., Huang R., Brecher M., Koetzner C.A., Zhang J., Chen H., Qin C.F., Zhang Q.Y., Zhou J., Kramer L.D., Xia M, & Li H. (2017). Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease. Antiviral research, 150, 217-225.

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Quantifying ChIP-seq Enrichment by qPCR

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ChIP-ed samples and their respective input controls were assessed by qPCR at four selected loci using primers listed in Supplementary Table S2. The primer sets and exponential amplification range were assessed to ensure amplification efficiencies around 100% (±10%). For each reaction, 0.5 μl of 10 μM forward and reverse primers were mixed with 10 μl of 2× Power SYBR-green master mix (Applied Biosystems, Waltham, MA) and diluted to a total volume of 19 μl with RNase-free H₂O. One μl of ChIP-ed DNA or input controls (diluted 100-fold to reach concentrations between ∼0.1–5 ng/μl) were added to each well and mixed in the MicroAMP fast optical 96-well reaction plate (Thermo Fisher Scientific, Waltham, MA). The loaded plate was amplified for 40 cycles via the ViiA 7 real-time PCR system (Thermo Fisher Scientific, Waltham). For each biological replicate (n = 3), three technical triplicates were performed. The fold enrichment was calculated as: where Ct, subscript IC and subscript ChIP, represent cycle threshold values, input controls, and ChIP-ed DNA, respectively, whereas trkH was used as an unenriched control site for normalization.

Tang J, & Brynildsen M.P. (2023). Genome-wide mapping of fluoroquinolone-stabilized DNA gyrase cleavage sites displays drug specific effects that correlate with bacterial persistence. Nucleic Acids Research, 51(3), 1208-1228.

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Differential Scanning Fluorimetry of Proteins

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Differential scanning fluorimetry (DSF) was performed using a 7500 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA) by selecting the ROS filter. A master mix solution was prepared with 0.133 mg mL -1 of protein and 1:500 of SYPRO Orange (ThermoFisher Scientific, Waltham, MA, USA) in 50 mM Sodium Acetate pH 5.0. 15 μL of this mixture were added to 5 μL of buffer to compare wtCP with mutants, and 5 μL of different concentration of sugar polymers in a MicroAmp Fast Optical 96-Well Reaction Plate (# 4346906), sealed with MicroAmp Optical Adhesive Film (all from ThermoFisher Scientific, Waltham, MA, USA) in a final volume of 20 μL for each measurement. All samples were prepared in duplicate. The samples where heated with a ramp speed that provides a 2 min pause at 35 °C, followed by a ramp to 95 °C over 90 min. The SYPRO Orange fluorescence was plotted versus temperature (melting curve) and the resulting plot converted into its first order derivative, which provided the melting temperature (T m ) as a positive peak.

Luti S., Bemporad F., Vivoli Vega M., Leri M., Musiani F., Baccelli I, & Pazzagli L. (2020). Partitioning the structural features that underlie expansin-like and elicitor activities of cerato-platanin. International journal of biological macromolecules, 165(Pt B).

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Quantitative Gene Expression Analysis

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Total RNA was isolated from WT and LiHsp83-SAG1 leaves using Trizol reagent (Invitrogen), following the manufacturer’s instructions. The RNA concentration and its integrity were analyzed as previously [17 (link)]. cDNA was synthesized using oligo dT₂₀ (Invitrogen) and M-MLV reverse transcriptase (Promega) according to the manufacturer’s instructions. This cDNA was used as a template for Real-Time quantitative PCR (qRT-PCR). The steady-state mRNA levels were analyzed by qRT-PCR as indicated previously [18 (link)]. Primer sequences for all the experiments are listed in Supplementary Table S1. Relative quantification was performed by the comparative cycle threshold method. The elongation factor alpha from Nicotiana tabacum gene (NtEFα) was used as endogenous control. Reactions were carried out in MicroAmp™ Fast Optical 96-Well Reaction Plate (Thermo Scientific) using the StepOnePlus Real-Time PCR System and the Mx3005P qPCR Software 4.0 (Stratagene). For comparative purposes, relative gene expression was defined with the value of −Log₂ in each control plants.

Corigliano M.G., Albarracín R.M., Vilas J.M., Sánchez López E.F., Bengoa Luoni S.A., Deng B., Farran I., Veramendi J., Maiale S.J., Sander V.A, & Clemente M. (2019). Heat treatment alleviates the growth and photosynthetic impairment of transplastomic plants expressing Leishmania infantum Hsp 83-Toxoplasma gondii SAG1 fusion protein. Plant science : an international journal of experimental plant biology, 284, 117-126.

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RNA Extraction and qPCR Analysis of Cytokine Expression

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As described previously [61 (link)], total RNA was extracted from ±30 mg snap-frozen LV tissue using RNeasy fibrous tissue kit (Qiagen, Venlo, The Netherlands) following the manufacturer’s guidelines. The concentration and purity of RNA were assessed with the NanoDrop 2000 spectrophotometer (Isogen Life Science, Utrecht, The Netherlands). RNA was reverse-transcribed to cDNA using the qScript cDNA SuperMix (Quantabio, VWR, Leuven, Belgium). The expressions of interferon-γ (IFN-γ) and interleukin-6 (IL-6) were studied. Primers (Table S2) were designed in the coding sequence of the mRNA. Real-time PCR was carried out in a MicroAmp Fast Optical 96-well reaction plate (Thermo Fisher Scientific, Merelbeke, Belgium) using the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). SYBR Green (Thermo Fisher Scientific) chemistry-based qPCR was performed [62 (link)]. Gene expression data were analyzed via the ΔΔCt method with consideration of the MIQE guidelines [63 (link)]. The most stable reference genes for this experimental set-up were determined by geNorm (hypoxanthine-guanine phosphoribosyl transferase (HPRT) and phosphoglycerate kinase 1 (PGK1); Table S2).

Evens L., Beliën H., D’Haese S., Haesen S., Verboven M., Rummens J.L., Bronckaers A., Hendrikx M., Deluyker D, & Bito V. (2021). Combinational Therapy of Cardiac Atrial Appendage Stem Cells and Pyridoxamine: The Road to Cardiac Repair?. International Journal of Molecular Sciences, 22(17), 9266.

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