Single Cell Analysis and Other Fluidigm Services
With with Fluidigm's innovative microfluidic technology you can analyze single-cell or bulk-cell samples by:
- Real-Time PCR
- Digital PCR
- SNP Genotyping
- Library Preparation for Next-Generation Sequencing
Fluidigm Instruments Available Here:
- C1 Single-Cell Auto Prep System - The first commercially available, automated single-cell isolation and preparation system for genomic analysis. The C1 System provides an easy and highly reproducible workflow to process 96 single cells for DNA or RNA analysis.
- BioMark HD System - The BioMark Reader is a real-time PCR instrument designed to run Fluidigm Integrated Fluidic Circuits (IFCs) in either real-time or end-point read modes. The C1 Single-Cell Auto Prep System coupled with the BioMark HD System streamlines gene expression analysis through seamless effort and significant time savings with support for up to 96 individual cells across 96 transcripts.
- Juno (Coming Soon!) - Juno combines thermal cycling and IFC control to enable hands-free genotyping from low sample amounts-only 6.9 ng of DNA is needed! With the Juno 96.96 Genotyping IFC you can analyze 96 SNPs in 96 samples to get 9,216 data points in just under three hours. The Juno system can also load all gene expression and digital PCR IFCs, which makes it the most versatile IFC controller Fluidigm has to offer.
C1 Integrated Fluidic Circuit (IFC)
How Does it Work?
1) Up to 96 single-cells are captured on a C1 Integrated Fluidic Circuit (IFC) on the C1 Single-Cell Auto Prep System. Single-cells are then lysed in individual microfluidic chambers on the IFC and undergo reactions such as reverse transcription and DNA amplification, which will vary depending on your downstream analysis.
2) Samples are harvested from the C1 IFC and transferred to a 96-well plate. These samples can be single-cell cDNA or genomic DNA that has undergone amplification. If you are preparing libraries for Illumina Sequencing, then you stop here and proceed with manual library preparation.
3) Samples are transferred to any one of a variety of Fluidigm's gene expression or genotyping IFCs along with the desired assays. The samples and assays are "loaded" by an IFC controller, such as the Juno system, and then analyzed on the BioMark HD System. These samples could be either single-cell samples harvested from the C1 or bulk cell cDNA or genomic DNA samples obtained through more conventional methods. BioMark IFCs come in several formats to give you flexibility in your experimental design (see table below). For example, in a 48.48 IFC, 48 samples are ran against 48 assays, such that 2,304 independent reactions are processed in parallel (not multiplexed) in nano liter reaction chambers.
Schematic of a BioMark 48.48 Integrated Fluidic Circuit (IFC)
IFC Options Plate Format No. of Samples No. of Assays Reactions per Run Flex Six 12 12 144 48.48 48 48 2,304 96.96 96 96 9,216 192.24 192 24 4,608
Pricing Information* Service Cost Cell QC (check cell size, viability, and buoyancy) $90 per cell type Single Cell Capture on the C1 for qPCR $700 per capture (up to 96 cells) Single Cell Capture on the C1 for mRNA-seq $1,800 per capture (up to 96 cells) qPCR on the BioMarK with a 96.96 IFC $1,400 per run (9,216 reactions) Single-Cell Library Prep for mRNA-seq $1,400 per 96 single-cell samples Single-Cell Library Prep for mRNA-seq $900 per 48 single-cell samples
- *Prices are estimates and do not encompass all the Service provided by CGF. Please contact CGF for a quote.
If you are interested in single-cell analysis or using the Juno, C1, or BioMark systems, contact us for more information.
Genome-Wide Expression Profiling by Microarray
Affymetrix Array Probe Distribution
Affymetrix GeneChips are the most commonly used microarray platform for genome-wide expression profiling. Affymetrix offers several different whole genome arrays for expression analysis with varying levels of coverage:
- 3' Arrays, such as the PrimeView Array, use probe sets targeted to the 3’ end of polyadenylated transcripts and have an emphasis on established, well annotated content.
- Whole-transcript Arrays, such as the Gene and Exon Arrays, use probes that target the entire length of the transcript instead of just the 3’ end. Whole-transcript arrays are also not limited to polyadenylated transcripts, enabling the interrogation of non-coding RNA.
- Gene Arrays use approximately 20 probes per transcript, which are summarized into a single expression value representing all transcript isoforms from the same gene, providing investigators with an accurate picture of overall gene expression.
- Exon Arrays provide higher resolution coverage than Gene arrays with approximately four probes per exon and 40 probes per transcript allowing for exon-level expression profiling for alternative splicing events as well as gene-level expression.
- Transcriptome Arrays offer unparalleled coverage of both coding and non-coding transcripts, with data quality equal to two full lanes of next-generation sequencing. With approximately ten probes per exon and four probes per exon-exon splice junction, Transcriptome Arrays provide the increased coverage required to accurately detect all transcript isoforms produced by a gene. 70% of the probes on this array cover exons for coding transcripts, and the remaining 30% of probes on the array cover exon-exon splice junctions and non-coding transcripts.
- miRNA Arrays profile small non-coding RNA (miRNA, snoRNA, and scaRNA) in well over a hundred organisms and provides 100% coverage of mature miRNA sequences in version 20 of miRBase.
We will provide:
- RNA extraction/purification
- Sample QC
- Amplification and probe labeling
- GeneChip array hybridization
- GeneChip array scanning and data analysis.
For typical sample processing, we require at least 200 ng of total RNA in water with a concentration no less than 50 ng/ul. If you have a very small amount of RNA, we can process as little as 0.1 ng of RNA for microarray analysis. Please refer to our sample submission page for more details.
Samples delivered for microarray analysis are usually completed and returned with an analysis report in 3-4 weeks.
Pricing Information* Service Organism(s) Price/Sample Expression Profiling with Transcriptome Arrays Mo $370 Expression Profiling with Transcriptome Arrays Hu $270** Expression Profiling with Exon 1.0 ST Arrays Hu $250** Expression Profiling with Gene 2.0 ST Arrays Mo, Rat $330 Expression Profiling with Gene 2.0 ST Arrays Hu $200** Expression Profiling with Gene 1.0 ST Arrays Hu $150** Expression Profiling with Gene 1.0 ST Arrays other inquire Expression Profiling with PrimeView Arrays Hu $270 miRNA Profiling with miRNA 2.0 Arrays over 100 $150** miRNA Profiling with miRNA 4.0 Arrays over 200 $300
- *Prices include cost of labor, reagents, and arrays. Increase price by $100/sample for FFPE samples or for very low amounts of RNA input.
- **Prices are good while supplies last.
Genome-Wide Expression Profiling by RNA-seq
We provide services for whole transcriptome, mRNA, and 3' RNA sequencing on Illumina platforms. Please contact us for more details.
SNP genotyping with the Sequenom MassARRAY System is the most economical method for analyzing a targeted set of SNPs in a large number of samples and is ideal for the validation of GWAS studies, as well as genetic testing of SNP/mutation panels of interest. The system allows for flexible experimental design and is scalable for the analysis of a few to hundreds of SNPs over multiple samples.
Sequenom also offers a several pre-designed assays for mutation profiling ideal for cancer reaserch, including OncoCarta™ Panels (v1.0, v2.0, v3.0), for comprehensive mutation screening of solid tumors, as well as a tissue-specific lung adenocarcinoma panel, LungCarta™ Panel, and a tissue-specific melanoma panel, MelaCarta™ Panel v1.0. Somatic profiling with the MassARRAY Analyzer 4 System makes it possible detect mutation frequencies as low as 5% in the original tumor-derived template DNA.
Sequenom SNP genotyping uses primer-extension chemistry to generate allele-specific products with distinct masses for detection with Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry. Allele discrimination is conferred by a single-base primer extension across the SNP site with a mass-modified dideoxynucleotide terminator. The mass differences between each of the four modified terminators is sufficiently large to be detected by the MassARRAY System. Starting with the known molecular mass of the input extension primers and the four terminators, the data analysis software can determine which terminator was added to each extension primer and thus assign the nucleotide in the SNP position.
Estimated Price per Project* 5 SNPs 35 SNPs 70 SNPs 105 SNPs 140 SNPs 50 Samples $1,200 $1,600 $2,200 $2,800 $4,000 100 Samples $1,200 $1,600 $2,800 $3,400 $4,600 200 Samples $1,700 $2,200 $3,400 $4,600 $5,800 400 Samples $2,300 $2,800 $4,500 $6,300 $8,000 600 Samples $2,900 $3,300 $5,700 $8,000 $10,400 800 Samples $3,500 $3,900 $6,800 $9,800 $12,700 1000 Samples $4,000 $4,500 $8,000 $11,500 $15,000 Estimated Price per Assay 5 SNPs 35 SNPs 70 SNPs 105 SNPs 140 SNPs 50 Samples $4.80 $0.91 $0.63 $0.53 $0.57 100 Samples $2.40 $0.46 $0.40 $0.32 $0.33 200 Samples $1.70 $0.31 $0.24 $0.22 $0.21 400 Samples $1.15 $0.20 $0.16 $0.15 $0.14 600 Samples $0.97 $0.16 $0.14 $0.13 $0.12 800 Samples $0.88 $0.14 $0.12 $0.12 $0.11 1000 Samples $0.80 $0.13 $0.11 $0.11 $0.11
- *The cost varies significantly from project to project, and these prices are only rough estimates. Please contact CGF for a quote.
Promoter and Methylation Analysis
Targeted Methylation Analysis with the Sequenom MassARRAY System
Sequenom’s MassARRAY EpiTYPER application is capable of high-throughput detection and quantification of DNA methylation at one or many genomic sites of interest. This method relies on bisulfite-treatment of DNA, which converts non-methylated cytosine into uracil while leaving methylated cytosine unchanged. Each reaction assays all the CpG positions in a 200-600 bp amplification product of bisulfite-treated DNA. The EpiTYPER software generates a report of the methylation status of the original sample including the ratio of methylated to non-methylated DNA with detection of methylation levels as low as 5%.
Genome-Wide Gene Regulation Analysis with Affymetrix Tiling Arrays
As opposed to expression arrays where probes are focused to known or predicted transcripts, tiling arrays sequentially probe the entire genome in an unbiased fashion. Tiling arrays are most commonly used to map sites of protein/DNA interaction in ChIP-on-chip experiments, but can also be used for methylation mapping (MeDIP-chip), nucleosome mapping (DNase-chip), and discovering novel RNA transcripts.
Affymetrix's Human Promoter 1.0R Array or Mouse Promoter 1.0R Array is ideal for most ChIP-on-chip experiments and provides high-resolution coverage in over 25,500 human promoter regions. Probes are tiled at an average resolution of 35 bp, as measured from the central position of adjacent 25-mer oligos, leaving a gap of approximately 10 bp between probes. Each promoter region covers approximately 7.5 kb upstream through 2.45 kb downstream of 5' transcription start sites. For over 1,300 cancer-associated genes, coverage of promoter regions was expanded to include additional genomic content. This more extensive coverage spans from 10 kb upstream through 2.45 kb downstream of transcriptional start sites.
We charge $420/sample for analysis on the Human Promoter 1.0R Arrays, which includes the cost of labor, reagents, and the array. Please inquire for the pricing of other tiling arrays.
Cytogenetics is the study of chromosome structure, function, and composition and its role in heredity and disease. Chromosomal abnormalities such as aneuploidies, deletions, duplications, and translocations are the basis for many birth defects and syndromes and play an important role in cancer. Affymetrix’s CytoScan HD and OncoScan Arrays provide genome-wide detection of numerous types of chromosomal abnormalities in human samples. In addition to identifying copy number changes, these assays can detect loss of heterozygosity (LOH), uniparental isodisomy (UPD), low level mosaicism, and regions identical-by-descent.
The CytoScan HD Arrays require at least 250 ng of DNA and are compatible DNA extracted from fresh, or frozen tissues, as well as blood, saliva, and cultured cells. The OncoScan Arrays require only 80 ng of DNA and are specially designed for DNA extracted from FFPE tissue.
We charge $550/sample for cytogenetic analysis with the CytoScan Arrays, which includes the cost of labor, reagents, and the array. Please inquire for the pricing for OncoScan Arrays.
The figure to the right shows an example of CytoScan data in the Affymetrix Chromosome Analysis Suite Software illustrating a copy number of 1, 2, and 3 on chromosome 8, which was confirmed by FISH.
Bioinformatics Support and Access to Specialized Software
Laboratory will assist investigators with:
- Data analysis
- Assistance with the use of the available software for data analysis
Assistance With Experimental Design and Grant Submissions
Our staff is available for consultation to discuss experimental design and grant submission assistance. Letters of support or collaboration can be provided as requested.
A standard description of the RRC for inclusion in grant applications is available from the RRC web site. Please contact our staff about any additional information.
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