HT RT-qPCR was run on the BioMark HD System, using 96 × 96 Fluidigm Dynamic Arrays (Cat. #BMK-M-96.96; Fluidigm, South San Francisco, CA). Preparation and loading of Fluidigm 96.96 Dynamic Array IFC (integrated fluidic circuit) were performed according to the manufacturer's instructions. Preparation of the 96.96 Dynamic Array IFC included the injection of 150 µL of a control line fluid into each chip accumulator with a syringe. The chip was then placed into the Juno and run with the prime 96.96 GE. After priming, the chip was loaded with samples, and the primer reaction was mixed within 1 h to reduce the pressure loss within the chip. Thus, 5 µL of each primer reaction mix and each sample were loaded into respective inlets. Samples and primer reaction mixes were loaded into the chip into the Juno and running the Load mix 96.96 GE script. The chip was transferred into the BioMark™ HD System, and qPCR and melting curve analysis were performed by running the following temperature program: 2400 s at 70 °C and 30 s at 60 °C, followed by a hot start for 60 s at 95 °C, 30 PCR cycles of 5 s at 96 °C for denaturation and 20 s at 60 °C for annealing and elongation. The melting curve analysis consisted of 3 s at 60 °C followed by heating up to 95 °C with a ramp rate of 1 °C/3 s.
Fluidigm 96.96 dynamic array
Manufactured by Standard BioTools
Sourced in United States
The Fluidigm 96.96 Dynamic Arrays is a specialized lab equipment designed for high-throughput sample and assay preparation. It enables the simultaneous analysis of up to 9,216 individual real-time PCR reactions in a single experiment.
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Lab products found in correlation
Biomark hd system, by Standard BioTools (6 mentions)
Rneasy 96 qiacube ht, by Qiagen (3 mentions)
Fluidigm real time pcr analysis 2, by Standard BioTools (3 mentions)
Taqman primer, by Thermo Fisher Scientific (2 mentions)
Taqman preamplification master mix, by Thermo Fisher Scientific (2 mentions)
Biomark system, by Standard BioTools (1 mentions)
Taqman 2x universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Real time pcr analysis software, by Standard BioTools (1 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin tissue kit, by Macherey-Nagel (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna kit, by Thermo Fisher Scientific (1 mentions)
Taqman preamp master mix, by Thermo Fisher Scientific (1 mentions)
14 protocols using fluidigm 96.96 dynamic array
1
High-Throughput RT-qPCR on Fluidigm BioMark HD
2
Fluidigm Dynamic Array-based qPCR
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Samples and assays (primer pairs) were prepared for 96.96 Fluidigm Dynamic arrays (Fluidigm, San Fransisco, CA, USA) according to the manufacturer’s recommendation. Briefly, assays were prepared by dispensing 20X Fluidigm Gene Expression assay Loading Reagent and the matching 20X TaqMan Gene Expression Assay into the wells of a 10X Assay Plate. Samples were mixed with 20X Fluidigm Sample Loading Reagent and TaqMan Universal PCR Master Mix 2X (Applied Biosystems). A 96.96 Dynamic Array primed chip was then loaded with assays and samples, and the real time-PCR was run on a Biomark System for Genetic Analysis according to the Fluidigm Protocol. All reactions were made in duplicate. Data were collected and analyzed using Fluidigm Real-Time PCR Analysis software.
3
Fluidigm-based Gene Expression Analysis
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Reverse transcription was performed using 50 ng of RNA with a Reverse Transcription kit (Thermo Fisher Scientific) and cDNA was preamplified (14 cycles) using a pool of TaqMan primers, following the manufacturer's instructions (Fluidigm). Sample and assay preparation of the 96.96 Fluidigm Dynamic arrays were carried out according to the manufacturer's instructions. Data were collected and analyzed using Fluidigm Real-Time PCR Analysis 2.1.1 software (Fluidigm Real-time PCR Analysis software, RRID:SCR_015686) and Ct values used to calculate ddCt and fold change. Significant P values were calculated by performing a Student t test on the -ddCt values. Signature scores were calculated per sample as the mean of the expression of the genes (-dCt) in each signature and P values calculated using a two-sample t test. Data was plotted using TIBCO Spotfire software (RRID:SCR_008858).
4
Quantitative Gene Expression Analysis
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Total RNA was isolated from snap frozen tissue and cells using Qiashredder and Qiazol Lysis Buffer on Qiacube-HT following the RNeasy 96 QIAcube HT total RNA cell with DNase protocol according to manufacturer’s instructions (Qiagen). Reverse transcription was performed from 50 ng of total RNA (Thermo Scientific #4374967) and genes of interest were pre-amplified (Thermo Scientific #4488593; 14 cycles) using a pool of TaqMan primers (listed in Additional file 8 : Table S3), following the manufacturer’s instructions (Thermo Scientific), and further run on a 96.96 Fluidigm Dynamic array on the Biomark according to the manufacturer’s instructions (Fluidigm). Data was collected and analyzed using Fluidigm Real-Time PCR Analysis 2.1.1 providing Ct values. All gene expression calculations were performed in Jmp®13.0.1, and data represented in TIBCO Spotfire® 6.5.2 or GraphPrism®. Ct values were normalized to the average of housekeeping genes (dCt), and all treatment group compared to the average control group (-ddCt) and Fold Change was calculated by taking 2^-ddCt. Statistical analysis of gene expression data (-ddCt) was performed in Jmp®13.0.1, using a pairwise Student’s t-test, which identifies genes significantly modulated compared to control. GSVA scoring [32 (link)] was performed using genes defined in Rooney et al. [33 (link)] (Additonal file 8 : Table S3).
5
Gene Expression Analysis of Snap-Frozen Tissues
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Total RNA was isolated from snap frozen tissue and cells using Qiashredder and Qiazol Lysis Buffer on Qiacube-HT following the RNeasy 96 QIAcube HT total RNA cell with DNase protocol according to manufacturer's instructions (Qiagen). Reverse transcription was performed from 50ng of total RNA (Thermo Scientific #4374967) and further pre-amplified (Thermo Scientific #4488593; 14 cycles) using a pool of TaqMan primers (listed in Table S1), following the manufacturer's instructions (Thermo Scientific), and further run on a 96.96 Fluidigm Dynamic array on the Biomark according to the manufacturer's instructions (Fluidigm). Data was collected and analysed using Fluidigm Real-Time PCR Analysis 2.1.1 providing Ct values. All gene expression calculations were performed in Jmp®13.0.1, and data represented in TIBCO™ Spotfire® 6.5.2 or GraphPrism®. Ct values were normalised to the average of housekeeping genes (dCt), and all treatment group compared (subtracted) to the average control group (-ddCt) and Fold Change was calculated by taking 2-ddCt. Statistical analysis of gene expression data (-ddCt) was performed in Jmp®13.0.1, using a pairwise Student's t-test, which identify genes significantly modulated compared to control.
6
Comprehensive RNA Isolation and Analysis
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Total RNA was isolated from snap-frozen tissue and cells using Qiashredder and Qiazol Lysis Buffer on Qiacube-HT following the RNeasy 96 QIAcube HT total RNA cell with DNase protocol according to the manufacturer's instructions (Qiagen). Reverse transcription was performed from 50 ng of total RNA (Thermo Fisher Scientific #4374967) and genes of interest were preamplified (Thermo Fisher Scientific #4488593; 14 cycles) using a pool of TaqMan primers (listed in Supplementary Table S1), following the manufacturer's instructions (Thermo Fisher Scientific), and further run on a 96.96 Fluidigm Dynamic array on the Biomark according to the manufacturer's instructions (Fluidigm). Data were collected and analyzed using Fluidigm Real-Time PCR Analysis 2.1.1 providing Ct values. All gene expression calculations were performed in Jmp13.0.1, and data represented in TIBCO Spotfire 6.5.2 or GraphPrism. C t values were normalized to the average of housekeeping genes (dC t ), and all treatment group compared with the average control group (ÀddC t ) and fold Change was calculated by taking 2^ÀddC t . Statistical analysis of gene expression data (ÀddC t ) was performed in Jmp13.0.1, using a pairwise Student t test, which identify genes significantly modulated compared with control. GSVA scoring (19) was performed using genes defined in Rooney and colleagues (20) .
7
Multiplexed qRT-PCR Assay for Placental Gene Expression
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Oligonucleotide primer sequences were designed using the Universal ProbeLibrary Assay Design Center on the Roche Resource webpage that also identified the appropriate UPL probe sequences for each amplicon (for primer sequences, see Additional file 2 ). First-strand cDNA for 50 placenta samples (a subset of those analyzed by pyrosequencing) was generated using supplier’s protocols for 50 ng of total RNA, which were subsequently pre-amplified (also referred to as specific target amplification; STA) for 14 cycles with a multiplexed pool of primers using the PreAmlification Master Mix (Fluidigm 100-5581). Subsequently, gene-specific RT-PCRs were performed with the same primers and specific UPLs on a Fluidigm 96.96 Dynamic Arrays using the Biomark HD system (Fluidigm Corp.) according to manufacturer’s instructions. The relative expression was quantified using the 2−ΔΔCt method and represented as fold change in gene expression normalised to the mean of the housekeeping gene RPL19 and the mean of all placentas. Only samples with two or more valid readings per triplicate were included.
8
High-Throughput RT-qPCR Analysis of Gene Expression
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RT-qPCR assays were performed using a microfluidic-based approach as previously described (Maupetit-Méhouas et al. 2016 (link)). First-strand cDNA was preamplified for 14 cycles with the pool of primers used for RT-qPCR and the TaqMan PreAmplification Master Mix (Life Technologies 4488593). Primer sequences are in Supplemental Table S6 . RT-qPCR assays were then performed and validated using Fluidigm 96.96 Dynamic Arrays and the Biomark HD system (Fluidigm) according to the manufacturer's instructions. The relative expression level was quantified with the 2-dCt. The housekeeping genes PPIA, TBP, and RPL13A were used to normalize transcript expression. Each analysis was performed in duplicate.
9
Chinook Salmon DNA Genotyping using SNPs
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Genomic DNA was isolated using a Macherey‐Nagel NucleoSpin® Tissue kit (Düren, Germany) following the protocols from the manufacturer. Some isolates from Chinook salmon carcasses contained low concentrations of DNA (<10 ng/μl), and these generally yielded low‐quality genotypes that were excluded from analyses. DNA was screened for a suite of 191 SNPs (Table S1 ) chosen from a larger database (288 SNPs) developed to coordinate genomic resources available for improving Chinook salmon fisheries management (Warheit et al., 2012 ). Exploratory analyses showed that different suites of SNPs had similar information content and performed equally well for fisheries applications (Warheit et al., 2012 ). Genotyping was performed on Fluidigm® 96.96 dynamic arrays under PCR conditions and concentrations recommended by Seeb et al. (2007 ), following a preamplification step by Smith et al. (2011 ).
10
Fluidigm-based qRT-PCR Gene Expression
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First-strand cDNA was pre-amplified for 14 cycles with the pool of primers used for the RT-qPCR analysis and the TaqMan PreAmplification Master Mix (Life Technologies, 4488593). RT-qPCRs were then performed and validated on Fluidigm 96.96 Dynamic Arrays using the Biomark HD system (Fluidigm Corp.) according to the manufacturer's instructions.
The relative gene expression was quantified using the 2−ΔΔCt method (35 (link)) that gives the fold changes in gene expression normalized to the geometrical mean of the expression of the housekeeping genes Arbp, Gapdh, Tbp and, according to the analysis, relative to one calibrator, as indicated in the text or figure legend. For each condition, the presented data were obtained from two independent experiments, each analyzed in duplicate.
The relative gene expression was quantified using the 2−ΔΔCt method (35 (link)) that gives the fold changes in gene expression normalized to the geometrical mean of the expression of the housekeeping genes Arbp, Gapdh, Tbp and, according to the analysis, relative to one calibrator, as indicated in the text or figure legend. For each condition, the presented data were obtained from two independent experiments, each analyzed in duplicate.
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