Expression level of 86 different microRNAs was determined by each chip containing of 94 samples in duplicate. Pre-amplified cDNA samples were diluted with low-EDTA (0.1 mmol/L) TE buffer (1:5). Almost 490 uL TagMan Universal PCR Master mix (Applied Biosystems) and 49 μL 20x GE Loading Reagent (Fluidigm, 85000746) were mixed and 3.85 μL of it were pipetted into a 96 well plate. Diluted pre-amplified cDNA were added to each well as 3.15 μL and then mixed then 5 μL of this mixture was loaded to inlets of a 96.96 Dynamic Arrays (Fluidigm). BioMark IFC controller HX (Fluidigm, San Francisco, CA) was used to deliver of both assay mix and sample mix from the loading inlets into the 96.96 Dynamic array reaction chambers for qRT-PCR by Fluidigm Integrated Fluidic circuit Technology. Cycling reaction condition in high throughput BioMark real time PCR followed as: first, thermal mix protocol followed by 50 °C for 2 min, 70 °C for 30 min and 25 °C for 5 min. Then UNG and Hot start protocol followed by 50 °C for 2 min and 95 °C for 5 min. Finally, PCR cycle followed by 40 cycles of 95 °C for 15 s (denaturation) and 60 °C for 60 s (annealing). Amplification curves, heat maps and cycle threshold (Ct) value were obtained by using real time qPCR analysis software. Linear baseline and threshold correction were performed automatically for entire chip.
96.96 dynamic array
Manufactured by Standard BioTools
Sourced in United States, Cameroon
The 96.96 Dynamic Arrays from Standard BioTools are a microfluidic device designed for high-throughput gene expression analysis. The device consists of a 96-well sample panel and a 96-assay panel, enabling the simultaneous processing of up to 9,216 individual gene expression reactions. The device utilizes integrated fluidic circuits to precisely control the flow and mixing of samples and reagents, allowing for efficient and accurate gene expression measurements.
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Lab products found in correlation
Taqman assay primer set, by Thermo Fisher Scientific (9 mentions)
Universal pcr master mix, by Thermo Fisher Scientific (6 mentions)
Cells direct, by Thermo Fisher Scientific (5 mentions)
Biomark hd system, by Standard BioTools (5 mentions)
Facsaria flow cytometer, by BD (4 mentions)
Dna suspension buffer, by Teknova (3 mentions)
Biomark hd, by Standard BioTools (3 mentions)
Snp genotyping analysis software version 4, by Standard BioTools (2 mentions)
Biomark real time pcr system, by Standard BioTools (2 mentions)
Tagman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Real time pcr analysis software, by Standard BioTools (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Biomark system, by Standard BioTools (2 mentions)
Hx ifc controller, by Standard BioTools (2 mentions)
Paxgene blood rna kit, by Qiagen (2 mentions)
Assay loading reagent, by Standard BioTools (2 mentions)
Dna binding dye sample loading reagent, by Standard BioTools (2 mentions)
Taqman preamp master mix, by Thermo Fisher Scientific (2 mentions)
Cellsdirect one step qrt pcr kit, by Thermo Fisher Scientific (2 mentions)
Taqman primer sets, by Thermo Fisher Scientific (1 mentions)
36 protocols using 96.96 dynamic array
1
High-throughput microRNA Expression Profiling
2
Flow Cytometry Sorting and qPCR Analysis of BM-MSCs
3
Dissociation and Single-Cell Analysis of Murine and Human Neuronal Cultures
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Embryonic and neonatal C57/bl6 brains (n = 3 mice per time point) were dissociated in papain solution for 5 min at room temperature, followed by the addition of inhibitor solution and gentle trituration to generate single-cell suspensions. Human iPSC-derived neuronal cultures at day 45 of differentiation from TS patients and control subjects (n = 3 lines each for controls and patients) were dissociated in accutase, and FACS sorting was performed at the Stanford shared FACS facility as previously described (Paşca et al., 2011 (link); Ho et al., 2018 (link)), excluding debris, dead cells and doublets to sort single cells into multi-well plates. sc-qPCR experiments were performed as in previous studies (Paşca et al., 2011 (link); Yoo et al., 2011 (link); Portmann et al., 2014 (link)) on 96 × 96 dynamic arrays (Fluidigm) and as recommended by the array manufacturer.
4
High-throughput qPCR of IFN-stimulated genes
5
Single-Cell Gene Expression Profiling of Adipose Stem Cells
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The S-ASCs and V-ASCs were obtained from the inguinal fat and omentum of 6 mice (C57BL/6, 3 months old, male) as previously described [6 (link)], all experiments being performed in accordance with the relevant guidelines and regulations. S-ASCs and V-ASCs were isolated from SVF (stromal vascular fraction) using the surface marker profile CD34+/CD73+/CD90+/CD105+ and CD45−/CD31− (to exclude hematopoietic and endothelial cell contamination). The cells were sorted as single cells using a Becton Dickinson FACSAria Flow Cytometer into a 96-well plate (one cell per well) with 6 μl lysis buffer in each well. Reverse transcription and low cycle preamplification were performed using CellsDirect (Invitrogen) with TaqMan primer sets (Applied Biosystems) as prescribed by the manufacturer. cDNA was loaded on 96.96 Dynamic Arrays (Fluidigm, South San Francisco, CA) for qPCR amplification using Universal PCR Master Mix (Applied Biosystems) with a specific TaqMan assay primer set [20 (link)].
6
Validating Drought-Associated SNPs
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For validation of drought susceptibility-associated SNPs, we conducted SNP genotyping on 96.96 Dynamic Arrays (Fluidigm) with integrated fluidic circuits (Wang et al., 2009 (link)) (N = 96) to validate the effectiveness of the identified SNPs in discriminating healthy from damaged trees. Prior to genotyping PCR, DNA extracts were normalised to approximately 5–10 ng/µl. They underwent a pre-amplification PCR (Specific Target Amplification [STA]) according to the manufacturer's protocol to enrich target loci. PCR products were diluted 1:10 with DNA suspension buffer (TEKnova, PN T0221) before further use. Genotyping was performed according to the manufacturer's recommendations. Four additional PCR cycles were added to accommodate for samples of lower quality or including inhibitors (von Thaden et al., 2020 (link)). Fluorescent data were measured using the EP1 (Fluidigm) and analysed with the SNP Genotyping Analysis Software version 4.1.2 (Fluidigm). The automated scoring of the scatter plots was checked visually and, if applicable, manually corrected.
7
Genome-wide and Targeted SNP Genotyping
8
Single-Cell Isolation and qPCR Analysis of ASCs
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ASCs isolated from freshly harvested WT, DM2, and DM1 SVF (obtained as described above) by using the surface marker profile CD45-/CD31-/CD34+ (to exclude contaminating CD45+ hematopoietic and CD31+ endothelial cells found within the SVF and to select for a putatively stem-like subset of CD34+ cells
[22 (link),24 (link)]) were analyzed and sorted as single cells by using a Becton Dickinson FACSAria flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) into 6 μL of lysis buffer. Propodium iodide exclusion was used to ensure that only live cells were sorted. Reverse transcription and low cycle pre-amplification were performed by using Cells Direct (Invitrogen) with TaqMan assay primer sets (Applied Biosystems) in accordance with the specifications of the manufacturers. cDNA was loaded onto 96.96 Dynamic Arrays (Fluidigm, South San Francisco, CA, USA) for qPCR amplification by using Universal PCR Master Mix (Applied Biosystems) with a uniquely compiled TaqMan assay primer set as previously described
[18 (link)].
[22 (link),24 (link)]) were analyzed and sorted as single cells by using a Becton Dickinson FACSAria flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) into 6 μL of lysis buffer. Propodium iodide exclusion was used to ensure that only live cells were sorted. Reverse transcription and low cycle pre-amplification were performed by using Cells Direct (Invitrogen) with TaqMan assay primer sets (Applied Biosystems) in accordance with the specifications of the manufacturers. cDNA was loaded onto 96.96 Dynamic Arrays (Fluidigm, South San Francisco, CA, USA) for qPCR amplification by using Universal PCR Master Mix (Applied Biosystems) with a uniquely compiled TaqMan assay primer set as previously described
[18 (link)].
9
Validating miRNA NGS Data by qPCR
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Individual small RNA samples from all groups were used for qPCR using the Fluidigm BioMark HD System to validate miRNA NGS data. Briefly, 250 ng of each small RNA sample was reverse transcribed using reverse transcription kit (Invitrogen) following the manufacturer's protocol. For specific target amplification, 5 μL pre-amplification sample mixture for each cDNA was prepared by mixing 2.5 μL PreAmp Master Mix, 1.25 μL of cDNA, 1 μL PreAmp Master Mix, 0.5 μL Pooled Delta Gene Assay Mix (500 nM) and 0.75 μL water. These reactions were incubated at 95 °C for 10 min, followed by 10 cycles of 15 s at 95 °C and 4 min at 60 °C, and then infinite hold at 4 °C. After incubation, the samples were cleaned using exonuclease I treatment method. Cleaned samples were diluted tenfold using DNA suspension buffer. Fluidigm quantitative measurement runs were carried out with 96.96 dynamic arrays (Fluidigm Corporation, CA, USA) according to manufactures instructions. The data were analyzed with real-time PCR analysis software in the BioMark HD instrument (Fluidigm Corporation, San Francisco, CA). 18S and SNORD21 were used as housekeeping controls. The correlation of the results between the two techniques was assessed using correlation coefficients of Pearson. The miRNA primers sequence was showed in supplementary (Table S1 ).
10
High-Throughput Microfluidic Real-Time PCR
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The BioMark™ real-time PCR system (Fluidigm, South San Francisco, CA, USA) was used for high-throughput microfluidic real-time PCR amplification using the 96.96 dynamic arrays (Fluidigm, South San Francisco, CA, USA). These chips dispensed 96 PCR mixes and 96 samples into individual wells, after which on-chip microfluidics assembled PCR reactions in individual chambers prior to thermal cycling resulting in 9216 individual reactions. Real-time PCRs were performed using FAM- and black hole quencher (BHQ1)-labeled TaqMan probes with TaqMan Gene Expression Master Mix in accordance with manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). Thermal cycling conditions were as follows: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 2-step amplification of 15 sec at 95 °C, and 1 min at 60 °C. Data were acquired on the BioMark™ real-time PCR system and analyzed using the Fluidigm real-time PCR Analysis software to obtain Ct values (see Michelet et al. 2014 for more details [10 (link)]). Primers and probes were evaluated for their specificity against cDNA reference samples. One negative water control was included per chip. To determine if factors present in the sample could inhibit the PCR, Escherichia coli strain EDL933 DNA was added to each sample as an internal inhibition control, using primers and probes specific for the E. coli eae gene [13 (link)].
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