384 well block module

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

The 384-Well Block Module is a component designed for use with various laboratory equipment and instrumentation. It provides a standardized platform for processing and handling microplates with 384 individual sample wells. The module's primary function is to facilitate efficient sample management and processing within compatible laboratory systems.

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9 protocols using 384 well block module

Quantification of Gene Expression in Mesenteric Arteries

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Total RNA was extracted from mesenteric arteries as previously described^{5 (link)}. Real time PCR was carried out on a 7900HT Fast Real-Time PCR machine with 384-Well Block Module (Applied Biosystems, Thermo Fischer Scientific, Paisley, United Kingdom) using gene-specific primers to quantify the relative abundance of each gene with SYBR Green I as the fluorescent molecule. The primers used were designed using the software Primer 3 and are listed in Electronic Supplemental Material Methods (Supplementary Table S1). PCR was performed in duplicate for each sample using a PCR Master mix for SYBR Green I (Applied Biosystems) containing 300 nmol/l primers, and 3 µl template cDNA in 10 µl total volume. PCR conditions consisted of an activation step of the Taq DNA polymerase (95 °C) for 10 min, followed by 40 cycles of 10 s at 95 °C (denaturation step) and 1 min at 60 °C (primer annealing, extension, and fluorescence acquisition). Serial dilution of pooled cDNA was used in each experiment to assess PCR efficiency. Ubiquitin C (Ubc) housekeeping gene was used as the reference gene for normalization. The relative copies number of the target genes were calculated with the 2^(−ΔΔCt) method, after assessment that PCR efficiency was 100%.

Nguyen Dinh Cat A., Callera G.E., Friederich-Persson M., Sanchez A., Dulak-Lis M.G., Tsiropoulou S., Montezano A.C., He Y., Briones A.M., Jaisser F, & Touyz R.M. (2018). Vascular dysfunction in obese diabetic db/db mice involves the interplay between aldosterone/mineralocorticoid receptor and Rho kinase signaling. Scientific Reports, 8, 2952.

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Quantitative RT-PCR for Gene Expression

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Using fresh hepatic tissue, RNA was isolated using a Qiagen RNeasy Plus Mini Kit (#74134), according to manufacturer’s instruction. RNA concentration was determined by optical density measurement at 260 nm on a spectrophotometer. RNA (1000 μg) was reverse-transcribed using an iScript^TM Reverse Transcription Supermix for RT-qPCR and a T100^TM Thermal Cycler (Biorad). 2 μL cDNA was used for the PCR reaction. The rt-qPCR was performed using the primers listed in Table S2C., Power SYBR^TM Green PCR Master Mix, and a 7900HT Fast Real-Time PCR System with 384-Well Block Module (Applied Biosystems^TM/ThermoFisher) running 40 cycles at 95°C for 15 seconds, 60°C for 60 seconds.

Shearn C.T., Fennimore B., Orlicky D.J., Gao Y.R., Saba L.M., Battista K.D., Aivazidis S., Assiri M., Harris P.S., Michel C., Merrill G.F., Schmidt E.E., Colgan S.P, & Petersen D.R. (2019). Cholestatic liver disease results increased production of reactive aldehydes and an atypical periportal hepatic antioxidant response.. Free radical biology & medicine, 143, 101-114.

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Genotyping of Alzheimer's Variants

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DNA was extracted from blood using AutoGenFlexStar (AutoGen) and FlexiGene Chemistry (Qiagen) or brain using AutoGen 245 T using standard protocols. Genotyping was performed using TaqMan assays (rs616338, C___2270073_20; rs72824905, C__97909430_10; rs429358, C___3084793_20; rs7412, C____904973_10) following manufacturers protocol, using a QuantStudio 7 Flex Detection System with a 384-Well Block Module (Applied Biosystems, Foster City, CA).

Conway O.J., Carrasquillo M.M., Wang X., Bredenberg J.M., Reddy J.S., Strickland S.L., Younkin C.S., Burgess J.D., Allen M., Lincoln S.J., Nguyen T., Malphrus K.G., Soto A.I., Walton R.L., Boeve B.F., Petersen R.C., Lucas J.A., Ferman T.J., Cheshire W.P., van Gerpen J.A., Uitti R.J., Wszolek Z.K., Ross O.A., Dickson D.W., Graff-Radford N.R, & Ertekin-Taner N. (2018). ABI3 and PLCG2 missense variants as risk factors for neurodegenerative diseases in Caucasians and African Americans. Molecular Neurodegeneration, 13, 53.

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Quantifying Notch Pathway Activation

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Total RNA was extracted using RNeasy Mini kit (QIAGEN) and 500ng of RNA was used for making cDNA using Reverse Transcription System (Promega). Each cDNA was analyzed in triplicate using real-time quantitative reverse transcription–PCR (qRT-PCR) assays in an ABI PRISM 7900HT sequence detection system with 384-well block module and automation accessory (Applied Biosystems). The information of the PCR primers and fluorogenic probes used are available on the Applied Biosystems website (GAPDH: Hs00266705, Notch1: Hs01062014_m1, Notch2: Hs01050702_m1, Notch3: Hs01128541_m1, Notch4: Hs00965889_m1). The relative expression mRNA level was normalized against the internal control GAPDH gene (ΔCt = Ct (target gene) – Ct (GAPDH)). The relative fold change was measured by 2-ΔΔCt formula compared to SUM159-Notch- control cells.

D’Angelo R.C., Ouzounova M., Davis A., Choi D., Tchuenkam S.M., Kim G., Luther T., Quraishi A.A., Senbabaoglu Y., Conley S.J., Clouthier S.G., Hassan K.A., Wicha M.S, & Korkaya H. (2015). Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity. Molecular cancer therapeutics, 14(3), 779-787.

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RNA Extraction and qRT-PCR Analysis

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Total RNA was extracted using RNeasy Mini or Micro kit (QIAGEN) and 1 μg of RNA was used for making cDNA using Reverse Transcription System (Promega). Each 20 μg of cDNA was analyzed in triplicate using real-time quantitative reverse transcription–PCR (qRT-PCR) assays in an ABI PRISM 7900HT sequence detection system with 384-well block module and automation accessory (Applied Biosystems).

Kim G., Ouzounova M., Quraishi A.A., Davis A., Tawakkol N., Clouthier S.G., Malik F., Paulson A.K., D’Angelo R.C., Korkaya S., Baker T.L., Esen E.S., Prat A., Liu S., Kleer C.G., Thomas D.G., Wicha M.S, & Korkaya H. (2014). SOCS3-mediated regulation of inflammatory cytokines in PTEN and p53 inactivated triple negative breast cancer model. Oncogene, 34(6), 671-680.

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RNA Extraction and qRT-PCR Analysis

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Quantitative PCR Analysis of Gene Expression

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Total RNA was isolated from cells using the NucleoSpin RNA kit (Machery-Nagel) according to the manufacturer’s instructions. RNA was quantified using a NanoDrop 2000c Spectrophotometer. cDNAs were synthesized from 2 μg of total RNA template using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). cDNA synthesis conditions were as follows: 25°C for 10 min, 37°C for 120 min, 85°C for 5 min. qPCR was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) on a 7900HT Fast Real-Time PCR System with a 384 well block module (Applied Biosystems). qPCR conditions were as follows: 50°C for 2 min, 95°C for 10 min, 95°C 15 s, 60°C for 1 min, with 40 cycles of amplification. The primers used for qPCR amplifications are shown in the Key resources table. Fold change mRNA expression levels were determined by the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The Ct value in each condition was normalized to an HPRT internal control and then normalized to siRNA vehicle control sample. Each cDNA was measured in triplicate per sample for each primer pair.

Wu I.H., Yoon J.S., Yang Q., Liu Y., Skach W, & Thomas P. (2021). A role for the ribosome-associated complex in activation of the IRE1 branch of UPR. Cell reports, 35(10), 109217.

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Quantitative PCR Enrichment Analysis

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Quantitative PCR was performed with the FastStart Universal SYBR Green Master Mix (Roche) using a 7900HT Fast Real-Time PCR System in a 384-Well Block Module (Applied Biosystems™). Primers used to detect enrichment at the INT sequence and at ACTA1 gene are listed in Supplementary Material, Table S4. Ct values were analysed using the SDS Software v2.4. The percentage of input reported was obtained by dividing the amount of precipitated DNA for the locus of interest by the amount in the input samples multiplied by 100%.

Yang B., Borgeaud A.C., Buřičová M., Aeschbach L., Rodríguez-Lima O., Ruiz Buendía G.A., Cinesi C., Taylor A.S., Baubec T, & Dion V. (2021). Expanded CAG/CTG repeats resist gene silencing mediated by targeted epigenome editing. Human Molecular Genetics, 31(3), 386-398.

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Genotyping of LBD and PSP Patients

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All control and a subset of 230 PSP (PSP Series 1) participants had genotypes obtained in our prior study [8 (link)]. We genotyped all 973 LBD-NP and an additional 810 PSP (PSP Series 2) patients using the same methods.
DNA was extracted from blood using AutoGenFlexStar (AutoGen) and FlexiGene Chemistry (Qiagen) or brain using AutoGen 245T using standard protocols. Genotyping was performed using TaqMan assays (ABI3_rs616338, C_2270073_20; PLCG2_rs72824905, C_97909430_10) following manufacturers’ protocol, using a QuantStudio 7 Flex Detection System with a 384-Well Block Module (Applied Biosystems, Foster City, CA).
All minor allele carriers (ABI3_rs616338-T, PLCG2_rs72824905-G) were confirmed using Sanger Sequencing. Polymerase chain reaction (PCR) primers with the following sequences were used to amplify and sequence the genomic region flanking the mutations: ABI3 5′-CTTCCTGCTCGCACCCGAC-3′, 5′-CTAATGCAGCATCCCCAACT-207 3′, PLCG2 5′- CCATAAATGAGGGCTCTCAG-3′, 5′-CATACCCACCTCACCCTTGT-3′. PCR products were purified using the Agencourt AMPure protocol (Beckman Coulter, CA) and sequenced using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing reactions were purified using Agencourt CleanSEQ (Beckman Coulter, CA) and run on an ABI33730xl Genetic Analyzer (Applied Biosystems). Sequences were analyzed using Sequencher 4.8 (Gene Codes Corporation, MI).

Strickland S.L., Morel H., Prusinski C., Allen M., Patel T.A., Carrasquillo M.M., Conway O.J., Lincoln S.J., Reddy J.S., Nguyen T., Malphrus K.G., Soto A.I., Walton R.L., Crook J.E., Murray M.E., Boeve B.F., Petersen R.C., Lucas J.A., Ferman T.J., Uitti R.J., Wszolek Z.K., Ross O.A., Graff-Radford N.R., Dickson D.W, & Ertekin-Taner N. (2020). Association of ABI3 and PLCG2 missense variants with disease risk and neuropathology in Lewy body disease and progressive supranuclear palsy. Acta Neuropathologica Communications, 8, 172.

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