Biomark hd platform

Manufactured by Standard BioTools

Sourced in United States

The Biomark HD platform is a high-throughput real-time PCR system designed for sensitive and accurate gene expression analysis. It enables simultaneous measurement of up to 9,216 genetic targets across 96 samples in a single run.

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9 protocols using biomark hd platform

Transcriptomic Analysis Using Fluidigm and 10x

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qRT-PCR analyses using the Fluidigm Biomark HD Platform was done as described (Säwén et al., 2016 (link)) (Supplementary file 2). PCA on gene expression data from all reference populations was performed using Clustvis, (http://biit.cs.ut.ee/clustvis/).
Single-cell RNA seq libraries were generated using a Chromium system (10x Genomics) according to the manufacturer’s instructions. Two consecutive sequencing runs were performed to achieve enough sequencing depth and data was combined and further analyzed using the Cell Ranger^TM pipeline (10x Genomics). The accession number for the single-cell RNA sequencing data reported in this paper is GSE122473.

Säwen P., Eldeeb M., Erlandsson E., Kristiansen T.A., Laterza C., Kokaia Z., Karlsson G., Yuan J., Soneji S., Mandal P.K., Rossi D.J, & Bryder D. (2018). Murine HSCs contribute actively to native hematopoiesis but with reduced differentiation capacity upon aging. eLife, 7, e41258.

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Real-Time PCR Quantification of Gene Expression

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MB 49 cells were homogenized immediately after detachment and RNA was extracted using the RNeasy Mini Kit (Qiagen) according to manufacturer instruction. The samples were stored at −80° C until reverse transcription PCR was performed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). No preamplification of cDNA products was performed. For real-time PCR, VeriQuest Probe qRT-PCR Master Mix (2X) (Affymetrix) and TaqMan^® Gene Expression Assays (Thermo Fisher Scientific) for each gene investigated, as well Actb and Gapdh as housekeeping genes, were purchased. Real-time PCR was performed on the Fluidigm Biomark HD platform on a 48.48 IFC chip with provided reagents and according to manufacturer’s instructions (User Guide: PN 68000089 H1). Real-time PCR results were analyzed in Real-Time PCR Analysis v4.3.1 software (Fluidigm). Gene expression levels were normalized to Gapdh (ΔC_t = C_tgene – C_tGapdh) and reported as relative mRNA expression (2^−(ΔCt)).

Oseledchyk A., Ricca J.M., Gigoux M., Ko B., Redelman-Sidi G., Walther T., Liu C., Iyer G., Merghoub T., Wolchok J.D, & Zamarin D. (2018). Lysis-independent potentiation of immune checkpoint blockade by oncolytic virus. Oncotarget, 9(47), 28702-28716.

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Single-Cell Gene Expression Analysis on Fluidigm Biomark HD

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For single-cell gene expression analysis using the Fluidigm Biomark HD platform, single cells were collected as described above in 5ul of 2X Reaction Mix. All validated TaqMan assays (i.e. 48 assays) were pooled to a final concentration of 0.2X (1:100 dilution) for cDNA synthesis and a target-specific pre-amplification step as previously described (Burton et al., 2013 (link)) on a MasterCycler system. 0.2μl of SuperScript III RT/Platinum Taq Mix, 2.5μl of the primer pool and 2.3μl of dH₂O were added to the 5μl Reaction Mix containing the single-cell sample. Cell lysis and cDNA synthesis were then performed at 50°C for 20 mins and the SuperScript III RT subsequ ently inactivated and Platinum Taq activated at 95°C for 2 mins. Pre-amplification of the specific targets was subsequently carried out with the following program: 95°C for 15 mins followed by 18 cycles of 60°C incubation for 4 mins. The resulting single-cell cDNA was diluted 5-fold in dH₂O and used for analysis in 48:48 dynamic array chips on a Biomark HD system (Fludigim) as per manufacturer’s specifications. Each single-cell sample cDNA was analyzed twice (technical duplicates).

Kang M., Garg V, & Hadjantonakis A.K. (2017). Lineage establishment and progression within the inner cell mass of the mouse blastocyst requires FGFR1 and FGFR2. Developmental cell, 41(5), 496-510.e5.

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Single-Cell Gene Expression Quantification

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Single cells were sorted into 5 μL of RT-STA mix directly for preamplification. The RT-STA mix contained 2× cells direct reaction mix (Invitrogen), 0.2 U/μL SUPERase (Ambion), 0.2× assay mix, 0.12× SuperScript III RT/Platinum Taq Mix (with RNaseOUT Ribonuclease Inhibitor) and topped up with Tris-EDTA (TE) buffer (Invitrogen). Gene expression quantification used the Biomark HD platform (Fluidigm). Gene filter procedure was as follows. For each gene, data with CtCall = FAILED and Ct_Quality < Ct_threshold were removed.

Xu X., Deobagkar-Lele M., Bull K.R., Crockford T.L., Mead A.J., Cribbs A.P., Sims D., Anzilotti C, & Cornall R.J. (2020). An ontogenetic switch drives the positive and negative selection of B cells. Proceedings of the National Academy of Sciences of the United States of America, 117(7), 3718-3727.

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Quantitative Gene Expression in Murine Cells

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CD71⁺ cells from murine bone marrow were sorted (100 cells/well) in 96-well plates containing 4ml/well of lysis buffer, and were snap frozen to −80°C. Control wells with 0, 20, 50 or 100 cells were included. Thawed plates were processed according to published protocol [23 (link)] where cDNA was pre-amplified in multiplex using gene specific Taqmanprimers and Taq/SSIII reaction mix (Invitrogen, Waltham, US), with an initial 50°C incubation step for the reverse transcription (RT). Gene expression analysis simultaneously on diluted samples was enabled using the 48.48 Dynamic array (Fluidigm, South San Francisco, US) and the Biomark HD platform (Fluidigm). The RT quantitative PCR (RT-qPCR) was run according to published protocol. RNA extraction was done using the RNeasy micro kit (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. Analysis was performed on a 7900 HT fast RT-PCR (ThermoFisher Scientific) and the primers used were Actin (#Mm02619580_g1), and HRI (#Mm01202300_m1), also from Thermi Fischer Scientific.

Wuyts J., Van de Peer Y., Winkelmans T, & De Wachter R. (2002). The European database on small subunit ribosomal RNA. Nucleic Acids Research, 30(1), 183-185.

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ChIP-seq Analysis of Pluripotency Factors

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ChIP assays using Otx2 (ProteinTech., 13497-1-AP), Oct4 (Oct-3/4; Santa Cruz, sc-8628), p300 (Upstate, 05-257), H3K27Ac (Abcam, ab8895), and H3K4me1 (Abcam, ab4729) were performed essentially as described previously (Lee et al. 2006 (link)) with 1 μg antibodies and 1 × 10⁶ ESCs for a transcription factor ChIP and 2.5 μg antibodies and 0.2 × 10⁶ ESCs for a histone ChIP. Bound regions were detected by quantitative PCR with the Fluidigm system. Prior to loading onto the BioMark HD platform (Fluidigm), ChIP samples were preamplified for 10 cycles with 2× TaqMan PreAmp Master Mix (Applied BioSystems).
For re-ChIP assays, 3 × 10⁶ cells were used with anti-Otx2 (first antibody) at 4°C overnight and continued as for the ChIP assay. After the 1× TE wash, immunoprecipitated complexes were twice eluted gently in 15 μl 10 mM dithiothreitol for 30 min at 37°C. Combined eluates were added to 1 ml IP buffer and incubated with the anti-Oct4 (second antibody) overnight at 4°C.

Yang S.H., Kalkan T., Morissroe C., Marks H., Stunnenberg H., Smith A, & Sharrocks A.D. (2014). Otx2 and Oct4 Drive Early Enhancer Activation during Embryonic Stem Cell Transition from Naive Pluripotency. Cell Reports, 7(6), 1968-1981.

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Comprehensive RNA Expression Analysis Using Fluidigm

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Cells from biological triplicates were harvested in TRIzol Reagent (LifeTechnologies) in weekly intervals and RNA was extracted according to the manufacturer’s instructions. Traces of contaminating DNA were removed using the TURBO DNA-free kit (LifeTechnologies). Synthesis of complementary DNA (cDNA) was performed using the SuperScript III First Strand Synthesis Mix (LifeTechnologies). For the non-enzyme control, a reference pool of all samples was used. cDNAs were subjected to specific target amplification prior to qPCR analysis using a pool of standard predesigned TAQman assays to be used later (Applied Biosystems) with a pre-amplification mastermix (Roche Life Science). Preamplified cDNA was diluted 1:5 and processed together with the TAQman assays for analysis in 96.96 Dynamic Array integrated fluidic chips on the BioMark HD platform (Fluidigm) according to the manufacturer’s instruction. Data were analyzed using Real-Time PCR Analysis software (Fluidigm).

Grabole N., Zhang J.D., Aigner S., Ruderisch N., Costa V., Weber F.C., Theron M., Berntenis N., Spleiss O., Ebeling M., Yeo G.W., Jagasia R, & Kiialainen A. (2016). Genomic analysis of the molecular neuropathology of tuberous sclerosis using a human stem cell model. Genome Medicine, 8, 94.

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FAIRE Profiling of Breast Tissue

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FAIRE was conducted as previously described [72 (link)]. In brief, breast tissue from 22 unrelated healthy female donors undergoing mammoplasty at Hannover Medical School was pulverized in liquid nitrogen and cross-linked with 1% formaldehyde followed by cell lysis. Chromatin was fragmented using the sonicator Bandelin Sonoplus HD 2070 to a final size of 200-400 bp. Input DNA was de-crosslinked by proteinase K incubation prior to phenol-chloroform extraction to isolate genomic DNA. FAIRE-enriched DNA was isolated first by phenol-chloroform extraction for the enrichment of open chromatin and incubated with proteinase K afterwards. DNA was precipitated by ethanol and prepared for genotyping via SNP-type assays (Fluidigm Inc.) using 48.48 Dynamic Genotypic IFC Arrays on the BioMark HD platform (Fluidigm Inc.) following the instructions of the manufacturer. SNP-type assays were custom designed by Fluidigm for SNPs 2, 3, 5 and 6. Cluster plots were evaluated for evidence of allelic imbalances in the FAIRE-enriched samples from individuals heterozygous for the variants of interest.

Helbig S., Wockner L., Bouendeu A., Hille-Betz U., McCue K., French J.D., Edwards S.L., Pickett H.A., Reddel R.R., Chenevix-Trench G., Dörk T, & Beesley J. (2017). Functional dissection of breast cancer risk-associated TERT promoter variants. Oncotarget, 8(40), 67203-67217.

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Comprehensive Cardiovascular Protein Profiling

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We determined 92 proteins related to immune regulation, metabolic pathways, and cardiovascular disease using a Proseek® multiplex platform (CVD II panel, Olink Proteomics, Uppsala, Sweden). Supporting Information, TableS1 lists the 92 biomarkers included in the CVD II panel. Fasting serum samples were analysed by the ARCADIA unit at the University Medical Center in Utrecht, the Netherlands. The platform applies proximity extension assay technology,⁸ where each protein gets linked to a unique pair of oligonucleotide ‐labelled antibodies. Next, hybridization, amplification, and subsequently quantification of the complementary oligonucleotide strands linked to the paired antibodies enable protein quantification by quantitative real‐time PCR using a Fluidigm BioMark HD platform. Quantitation data were quality controlled and normalized using internal and external controls, providing Normalized Protein eXpression (NPX) values. NPX is an arbitrary unit on a log2 scale used to quantify relative changes in protein levels. Higher NPX corresponds to higher protein expression. Five proteins were excluded due to bimodal distribution, leaving 87 proteins for analysis.

Cauwenberghs N., Sabovčik F., Magnus A., Haddad F, & Kuznetsova T. (2021). Proteomic profiling for detection of early‐stage heart failure in the community. ESC Heart Failure, 8(4), 2928-2939.

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