Fluidigm BioMark™ System was used according to the manufacturer’s instructions. High-quality total RNA (500 ng) (RIN > 7 on the Agilent Bioanalyzer) was reverse-transcribed using Invitrogen’s High Capacity cDNA Reverse Transcription Kits. The cDNAs were pre-amplified with a pool of 32 pairs of gene-specific primers for 14 cycles, followed by 40 cycles of real-time qPCR quantification in triplicates on 96.96 dynamic arrays using the Fluidigm gene expression protocol. Real-time PCR and data collection were done using the BioMark system, and data were analyzed with Fluidigm’s Real-Time PCR Analysis software. Affymetrix U133A array results were validated with measurements of the expression of 28 selected genes after treatment with 6 drugs for 24 hours in 24–60 cell lines (Supplementary Table S1 ).
Biomark system
Manufactured by Standard BioTools
Sourced in United States
The BioMark System is a high-throughput microfluidic platform designed for sensitive and accurate gene expression analysis. It enables parallel processing of multiple samples and target genes, facilitating efficient and automated data collection.
Automatically generated - may contain errors
Lab products found in correlation
Taqman preamp master mix, by Thermo Fisher Scientific (8 mentions)
Ssofast evagreen supermix with low rox, by Bio-Rad (7 mentions)
Real time pcr analysis software, by Standard BioTools (6 mentions)
Universal pcr master mix, by Thermo Fisher Scientific (6 mentions)
Taqman assay, by Thermo Fisher Scientific (6 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (6 mentions)
Biomark real time pcr analysis, by Standard BioTools (5 mentions)
Biomark real time pcr analysis software, by Standard BioTools (5 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (5 mentions)
Dna binding dye sample loading reagent, by Standard BioTools (5 mentions)
Assay loading reagent, by Standard BioTools (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Miscript 2 rt kit, by Qiagen (4 mentions)
Evagreen binding dye, by Biotium (4 mentions)
Qiashredder, by Qiagen (4 mentions)
Rt preamp master mix, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Miscript mirna assays, by Qiagen (3 mentions)
C1 single cell auto prep system, by Standard BioTools (3 mentions)
Cellsdirect one step qrt pcr kit, by Thermo Fisher Scientific (3 mentions)
84 protocols using biomark system
1
Fluidigm BioMark System for Gene Expression
2
Microfluidic qPCR Array for Splicing and Fatty Acid Metabolism
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A qPCR dynamic array based on microfluidic technology (Fluidigm, San Francisco, CA, USA) was used to simultaneously determine the gene expression of 14 components of the fatty acid metabolism, as well as of different components of the major spliceosome (n = 13), minor spliceosome (n = 4), and associated splicing factors (n = 14) in LNCaP and DU145 cell lines overexpressing miR-107 (Table S1 ). The expression of FASN was also determined in the prostate normal-like cell model (PNT2) in response to miR-107 overexpression. Moreover, the expression of three housekeeping genes (ACTB, GAPDH, and HPRT) was analyzed in the same samples. Specific primers for these transcripts were specifically designed with the Primer3 software and StepOne Real-Time PCR System software v.2.3 (Applied Biosystems, Foster City, CA, USA). Preamplification, exonuclease treatment, and qPCR dynamic array based on microfluidic technology were implemented as previously reported,49 (link),52 (link) following manufacturer's instructions, using the Biomark System and the Real-Time PCR Analysis Software (Fluidigm).
3
Gene Expression Analysis of Muscle Samples
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Total RNA was isolated from the Longissimus dorsi muscle of 114 samples with RiboPure kit (Ambion; Austin, TX, USA). Total RNA was quantified in a NanoDrop ND-1000 spectrophotometer (NanoDrop Products; Wilmington, DE, USA). The RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription (Applied Biosystems). The cDNA samples were loaded into a Dynamic Array 48.48 chip in a BioMark system (Fluidigm; San Francisco, CA, USA) through a NanoFlex controller following a protocol previously described18 (link). For this experiment, the expressed levels of 48 genes were analysed, and a total of 45 target genes were normalized using the two most stable housekeeping genes (ACTB and TBP). Primers used for the analyses are detailed in Supplementary Table S8 . Data were collected using the Fluidigm Real-Time PCR analysis software 3.0.2 (Fluidigm) and analysed using the DAG expression software 1.0.4.1157 (link), applying the relative standard curve method (See Applied Biosystems user bulletin #2). Analyses were performed using the normalized gene-expression levels of each sample and assay. The animals showing abnormal gene expression levels (outliers) were removed and data obtained were normalized if necessary by performing log2 transformation of the Normalized Quantity (NQ) value. The sex effect was also tested by using a linear model with R programme58 .
4
Isolation and Analysis of Engrafted Cells
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SCID mice were routinely euthanized at day 5 after surgery and border zones of hearts were removed and digested into single cells. The single cells were stained by H2-Kd and dyecycle (Invitrogen), and then subjected to FACS. MSCs isolated from donor transgenic mice were H2-Kd negative, whereas recipient SCID mice were H2-Kd positive. In addition, dyecycle staining was capable of distinguishing sing cell or fused cell via DNA content, and 2N was considered as a marker of single cell.
Inventoried TaqMan Assays (20×, Applied Biosystems) were pooled and diluted to a final concentration of 0.2× for each of the 24 probes. Individual cells were directly collected into 10 μl RT-PreAmp MasterMix (5.0 μl CellsDirect 2× Reaction Mix [Invitrogen]; 2.5 μl 0.2× assay pool; 0.5 μl RT/Taq enzyme [CellsDirect qRT-PCR kit, Invitrogen]; 2.0 μl TE buffer). The products were analyzed with Universal PCR Master Mix and inventoried TaqMan gene expression assays in 48.48 Dynamic Arrays on a BioMark System (Fluidigm, CA, USA). CT values were calculated from the system’s software (Fluidigm). The resulting values were then normalized to the endogenous controls by subtracting the average of GAPDH expression levels.
Inventoried TaqMan Assays (20×, Applied Biosystems) were pooled and diluted to a final concentration of 0.2× for each of the 24 probes. Individual cells were directly collected into 10 μl RT-PreAmp MasterMix (5.0 μl CellsDirect 2× Reaction Mix [Invitrogen]; 2.5 μl 0.2× assay pool; 0.5 μl RT/Taq enzyme [CellsDirect qRT-PCR kit, Invitrogen]; 2.0 μl TE buffer). The products were analyzed with Universal PCR Master Mix and inventoried TaqMan gene expression assays in 48.48 Dynamic Arrays on a BioMark System (Fluidigm, CA, USA). CT values were calculated from the system’s software (Fluidigm). The resulting values were then normalized to the endogenous controls by subtracting the average of GAPDH expression levels.
5
Single-Cell Transcriptome Profiling of Fetal Kidney
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Single cells were sorted by FACS into individual wells of 96 well plates. After
cells lysis, mRNA levels were measured by microfluidic single cell qPCR using
the Biomark system (Fluidigm, CA) according to the manufacturer’s
instructions. This resulted in 48 gene expression values (measured in threshold
cycles, Ct) for each one of the cells sorted. We analyzed approximately 160
cells from fetal human kidney after culturing for a single passage. qPCR
standard curves were created using serial dilutions of
“bulk” RNA containing a mixture of HeLa total RNA and
RNA from adult and fetal human kidneys. TaqMan gene expression primers and
probes were purchased from ThermoFisher Scientific. We clustered over the
following genes: NCAM1 (Assay ID Hs00941830_m1), PROM1 (CD133, Assay ID
Hs01009250_m1), and CDH1 (E-Cadherin, Assay ID Hs01023894_m1).
For clustering analysis, we standardized the expression levels of each gene
individually by subtracting the mean and dividing by 3 times the standard
deviation. Then, all values were truncated into the range [−1, +1].
Clustering was performed using complete linkage and correlation distance
(Matlab).
cells lysis, mRNA levels were measured by microfluidic single cell qPCR using
the Biomark system (Fluidigm, CA) according to the manufacturer’s
instructions. This resulted in 48 gene expression values (measured in threshold
cycles, Ct) for each one of the cells sorted. We analyzed approximately 160
cells from fetal human kidney after culturing for a single passage. qPCR
standard curves were created using serial dilutions of
“bulk” RNA containing a mixture of HeLa total RNA and
RNA from adult and fetal human kidneys. TaqMan gene expression primers and
probes were purchased from ThermoFisher Scientific. We clustered over the
following genes: NCAM1 (Assay ID Hs00941830_m1), PROM1 (CD133, Assay ID
Hs01009250_m1), and CDH1 (E-Cadherin, Assay ID Hs01023894_m1).
For clustering analysis, we standardized the expression levels of each gene
individually by subtracting the mean and dividing by 3 times the standard
deviation. Then, all values were truncated into the range [−1, +1].
Clustering was performed using complete linkage and correlation distance
(Matlab).
6
Real-Time PCR and Amplicon Sequencing
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Primers were designed with Primer360 (link). Real-time PCR was conducted using EvaGreen on the Biomark System (Fluidigm). PCR products from the same samples were mixed and barcodes were added. Finally, samples were pooled and sequenced using an Illumina MiSeq sequencer (see PCR experiment part in Supplementary material ).
7
Transcriptome Analysis of ccRCC and Metastasis
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Total RNA was isolated from fresh-frozen ccRCC and metastasis tissue using the mirVana™ miRNA Isolation Kit (Life Technologies) as previously described [18 (link), 19 (link)]. Genome-wide transcriptome analyses were performed using Human Transcriptome Array HTA 2.0 (Affymetrix) according to the manufacturer’s protocol. Further processing of microarray data were performed as previously described [18 (link)] (Additional file 1 : Supplementary methods). Gene expression data (generated using HuGene 1.0ST Affymetrix array) from 53 sunitinib-treated ccRCC patients were downloaded from ArrayExpress (E-MTAB-3267).
Quantitative real-time PCR (qRT-PCR) was performed using TaqMan technology on a BioMARK System (Fluidigm) as described previously [18 (link), 19 (link)]. TaqMan gene expression assays for 97 genes of the S3-score, as well as five genes used for normalization were purchased from Life Technologies. Further details about calculation of the S3-score based on interprofile correlations and development of a S3-score calculation model for use of qRT-PCR data are provided in the Additional file1 : Supplementary methods.
Quantitative real-time PCR (qRT-PCR) was performed using TaqMan technology on a BioMARK System (Fluidigm) as described previously [18 (link), 19 (link)]. TaqMan gene expression assays for 97 genes of the S3-score, as well as five genes used for normalization were purchased from Life Technologies. Further details about calculation of the S3-score based on interprofile correlations and development of a S3-score calculation model for use of qRT-PCR data are provided in the Additional file
8
MicroRNA Profiling in Clinical Samples
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After MicroRNAs were isolated from serum, saliva and tissue by MicroRNA isolation kit (Roche Diagnostics, GmbH, Mannheim, Germany) according to manufacturer's instructions; RNA samples converted to cDNA by using miScript II RT Kit (Qiagen). cDNA samples are preamplified by using miScript Microfluidics PreAMP Kit (Qiagen). qRT-PCR analysis performed by using miScript miRNA Assays (Qiagen) with Dynamic Array 96.96 (Fluidigm) on BioMark System (Fluidigm). Basically 95 types of microRNAs were studied.
9
Single-Cell qRT-PCR Using Fluidigm C1 and BioMark
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FACS-sorted single cells were captured using the C1 System (Fluidigm). Single-cell qRT-PCR was performed using wet-lab tested DELTAgene Assays (Fluidigm) in combination with EvaGreen chemistry using a BioMark System (Fluidigm). A complete list of primers used for this study is provided in Supplemental Experimental Procedures .
10
Quantitative RT-PCR for FLT1, GFP, and Gapdh
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Tissues were homogenized in TRIzol reagent with a homogenizer (Pro Scientific Inc., Oxford, CT, USA) immediately after tissue collection. Total RNAs were isolated using the QIAshredder (Qiagen, Valencia, CA, USA) and the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. Complementary DNAs were generated with SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative real-time RT-PCR assays were performed on the Biomark System (Fluidigm, San Francisco, CA, USA) using TaqMan assays (Applied Biosystems, Life Technologies Corporation, Foster City, CA, USA) for human FLT1 (Hs01052961_m1), GFP (Mr04097229_mr), and the mouse endogenous control gene Gapdh (Mm99999915_g1) according to the manufacturer’s recommendation.
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