BCH IDDRC Molecular Genetics Core Facility

Turnaround time: ~1 business day 

The Agilent 2100 Bioanalyzer system provides sizing, quantitation and quality control of DNA and RNA.

RNA Analysis

The Bioanalyzer RNA kits with the patented RNA Integrity Number (RIN)are the industry standard for RNA quality assessment, allowing reliable and precise integrity checks and sample quantitation before any RNA dependent application.

• Maximum flexibility – check total RNA, mRNA, or cRNA quality
• Minimal sample consumption – only 5 ng of total RNA or 50 pg of total RNA required for analysis
• RNA Integrity Number (RIN) – a reliable tool to compare

What the RIN can do:

• Obtain a numerical assessment of the integrity of RNA.
• Directly compare RNA samples, e.g. before and after archival, compare integrity of same tissue across different labs.
• Ensure repeatability of experiments, e.g. if RIN shows a given value and is suitable for microarray experiments, then the RIN of the same value can always be used for similar experiments given that the same organism/tissue/extraction method is used.

What the RIN cannot do:

• Tell a scientist ahead of time whether an experiment will work or not if no prior validation was done (e.g. RIN of 5 might not work for microarray experiments, but might work well for an appropriate RT-PCR experiment. Also, a RIN that might be good for a 3' amplification might not work for a 5' amplification).
• 100% guaranty a sample will amplify properly. There are some salts, phenols, and other contaminates that the Bioanalyzer cannot detect.  If these are present in a sample it may generate a good RIN but not amplify properly.

• RNA Integrity Database (RINdb) – a freely accessible repository holding hundreds of user submitted total RNA traces

Small RNA Analysis

The Agilent Small RNA kit provides a fast and sensitive analysis to resolve small nucleic acids in the size range of 6 to 150 nt.

• High resolution – miRNA smear region separated from tRNA
• Minimal sample consumption – use as little as 50 pg of purified miRNA or 10 ng of total RNA for analysis in 1 μL volume
• A faster and more sensitive alternative to agarose and polyacrylamide gels analysis for detection of small RNA
• Analyze total RNA (10-100 ng/μL), fractionated, or synthetic samples.

DNA Analysis

The DNA kits are ideal for automated sizing and quantitation of PCR fragments, restriction digests or fragmented DNA.

• Broad linear dynamic range – detection of less abundant products, e.g. non-specific amplifications
• High resolution – separate fragments down to 5 bp
• Sizing accuracy – normalization to internal markers and ladder
• Quantitation accuracy and reproducibility – quantitation of each DNA fragment against internal standards
• Minimal sample consumption – only 1 µL sample required

High Sensitivity DNA Analysis

The High Sensitivity DNA kit allows analysis of fragmented DNA or DNA libraries.

• Increased sensitivity - down to 5 pg/µL
• Analysis of complex DNA samples - down to 100 pg/µL for fragmented DNA or DNA libraries
• Broad linear dynamic range - detection of less abundant PCR products
• Smear analysis - specify size range of interest
• Quantitation accuracy and reproducibility - quantitation of each DNA fragment against internal standards
• Minimal sample consumption - only 1 µL of sample required

Free analysis software from Agilent: Agilent 2100 Expert Software

This is especially helpful with the DNA chips so you can perform the sizing to your specifcations. 

Customers must provide all reagents and slides. Core provides gaskets, wash buffers, and Human Cot-1.

The Agilent SureScan Scanner provides a precise and efficient microarray scanner system for many research applications. Designed for research using microarrays, the scanner system provides a laser-induced fluorescence scanning solution designed to read microarrays printed on standard 1 inch by 3 inch glass slides.

The microarray instrumentation measures the fluorescence intensity of two dyes simultaneously from labeled sample nucleic acid bound to microarrays. The system also enhances productivity with continuous loading from a 24-slide cassette and automatic loading of data files into Feature Extraction Software. It also features built-in ozone protection to minimize signal degradation.

QC analysis is provided by the core. In depth analysis can be done using the free Agilent analysis software for CGH & CGH+SNP Microarrays, CytoGenomics Software

Turnaround time: ~1 business day 

The 4200 TapeStation system separates nucleic acids by electrophoresis. The system detects:

• Fluorescently stained double-stranded DNA including genomic DNA
• Fluorescently stained total RNA (Eukaryotic and Prokaryotic)

Depending on your application, the software automatically determines the size, quantity, molarity, purity, RINe, DIN, %cfDNA, or ribosomal ratios. Reports can easily be generated and saved in PDF format, or exported for editing in Microsoft Word or Excel. 

Submit a 4ul aliquot of your sample for TapeStation assays.  The sample will not be returned.  Please list concentration values in iLab form.

Bio-Rad's QX200 droplet digital PCR system can obtain highly precise answers about genetic makeup. Absolute quantification is achieved by partitioning 20ul samples into 20,000 nanoliter-sized droplets, which allow for thousands of discrete measurements per sample. This massive sample partitioning is a key aspect of the ddPCR technique.

Following PCR amplification, droplets from each sample are analyzed individually on the QX200 Reader, where target-positive and negative droplets are counted to provide absolute quantification in digital form. The QX200 System works with both probe and EvaGreen fluorescence chemistry.

The ddPCR system can be used to:

• Detect rare DNA target copies (e.g. somatic mutations with low VAF)
• Determine copy number variation and gene expression with unmatched accuracy
• Precisely detect genome editing events
• Provide an absolute, standard-free measure of library concentration and quality, optimizing NGS performance overall

Available for full or self service. Contact Julia Phoon with questions/training requests: Julia.Phoon@childrens.harvard.edu

Helpful Links:

Planning Droplet Digital PCR Experiments

Droplet Digital PCR Applications Guide

Droplet Digital PCR Assay Finder or

PrimePCR Search Tools

QuantaSoft Analysis Pro Software- free analysis software

If you are unsure about what services you need or have a complex project, please click on the "contact the Core" tab at the top of the page and send us a message asking for clarification or a consultation.

Customers must provide all reagents and IFCs. Prices are subject to change depending on usage.

Regardless of chip format (48.48, 96.96, 192.24), samples should be submitted as follows:

TaqMan RNA Expression: At least 10ul of each cDNA sample (w/o preamp or 1:5 dilution of preamped) and 10ul of each assay (20X) in separate V-shape 96-well plates.

EvaGreen RNA Expression: At least 10ul of each cDNA sample (w/o preamp or 1:5 dilution of preamped) and 10ul of each primer pair (20uM) in separate V-shape 96-well plates.

When replicating samples or assays, please adjust the submission volume accordingly.

Data Deliverable

Your data will be available to download when a request is completed. Please go to "View My Requests" tab, click blue toggle icon to expand the request, and go to Attachments & URLs section, where you can find one or two zipped files (per chip), depending on the detection chemistry you choose. 

You will need the Fluidigm Real-Time PCR Software to analyze data for each service. User guides are also provided below. Please note that the software is designed to run on Windows machines only. For Mac users, please notify the core staff.

Real-Time PCR Analysis

Real-Time PCR Analysis User Guide

If you are unsure about what services you need or have a complex project, please click on the "contact the Core" tab at the top of the page and send us a message asking for clarification or a consultation.

Customers must provide all reagents and IFCs. Prices are subject to change depending on usage.

Regardless of chip format(48.48 or 96.96) you choose, samples should be submitted as follows:

TaqMan Genotyping: At least 10ul of each human gDNA sample at 60ng/ul* and 10ul of each assay (80X) in separate V-shape 96-well plates.

SNPtype Genotyping: At least 10ul of each human gDNA sample at 60ng/ul* and 10ul at 100 uM of each LSP, STA primer, and ASP1/ASP2 primer in separate V-shape 96-well plates.

 * The required concentration depends on the genome size of species.

When replicating samples or assays, please adjust the submission volume accordingly.

Data Deliverable

Your data will be available to download when a request is completed. Please go to "View My requests" tab, click blue toggle icon to expand the request, and go to Attachments & URLs section, where you can find one or two zipped files(per chip), depending on the detection chemistry you choose.

You will need Fluidigm Genotyping Software to analyze data from each service. User guides are also provided below. Please note that the software is design to run on windows machine only. For Mac users, please notify the Core staff. Exported excel file will be provided instead. If assistance is needed, please contact fluidigm@iddrc.org.

Genotyping Analysis

Genotyping Analysis User Guide

We will check sample quantity and quality for all full-service projects before we begin amplification.

Agilent Bioanalyzer

 We use the Agilent Bioanalyzer to check sample quality before starting amplification. The Bioanalyzer software automatically assigns an RNA Integrity Number (RIN) to a eukaryote total RNA sample. Using this tool, sample integrity is no longer determined by the ratio of the ribosomal bands, but by the entire electrophoresis trace of the RNA sample. This includes the presence or absence of degradation products. The assigned RIN is independent of sample concentration, instrument, and analyst, therefore becoming a de facto standard for RNA integrity.

RNA Integrity Number (RIN) – a reliable tool to compare samples

What the RIN can do:

• Obtain a numerical assessment of the integrity of RNA.
• Directly compare RNA samples, e.g. before and after archival, compare integrity of same tissue across different labs.
• Ensure repeatability of experiments, e.g. if RIN shows a given value and is suitable for microarray experiments, then the RIN of the same value can always be used for similar experiments given that the same organism/tissue/extraction method is used.

What the RIN cannot do:

• Tell a scientist ahead of time whether an experiment will work or not if no prior validation was done (e.g. RIN of 5 might not work for microarray experiments, but might work well for an appropriate RT-PCR experiment. Also, a RIN that might be good for a 3' amplification might not work for a 5' amplification).
• 100% guarantee that a sample will amplify properly. There are some salts, phenols, and other contaminants that the Bioanalyzer cannot detect.  If these are present in a sample it may generate a good RIN but not amplify properly.

 RNA Integrity Database (RINdb) – a freely accessible repository holding hundreds of user-submitted total RNA traces

Nanodrop

 We use the Nanodrop to check sample concentration and purity providing the sample is not below the detection limit of 2ng/ul. Using the 260/280 and 260/230 ratios we can help confirm sample purity.

• The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol, or other contaminants that absorb strongly at or near 280 nm.
• The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants that absorb at 230 nm, such as EDTA, carbohydrates, phenol, TRIzol, guanidine HCL, and guanidine isothiocyanate.

Full Service:

Samples should be prepared according to the following instructions and delivered to the Core Facility at CLS 16030.23. All samples for Affymetrix should be submitted in 1.5 mL centrifuge tubes labeled with the sample name or number including the following recommended amounts.

GeneChips for high quality/high concentration RNA – 100ng to 500ng of total RNA in 5ul, plus 2ul of total RNA (around 50ng/ul) for bioanalyzer QC.
GeneChips for lower quality(FFPE)/low concentration – 100pg to 10ng of total RNA in 5ul, plus 2ul of total RNA (around 50ng/ul) for bioanalyzer QC.

Submitting more than the required amount of samples is ok as we will normalize all your samples after we perform the QC.

GeneChip miRNA Arrays-

 Hyb-only Service:

GeneChip 3′ Expression  – 15ug biotinylated cRNA in less than 32ul.
GeneChip Exon, ST,HTA, Clariom Arrays – 10ug  of ssDNA( fragmented and terminal labeled) in 60ul.
GeneChip miRNA Arrays- 21.5ul biotin labeled sample

We are currently not running the CytoScan, OncoScan, or Karyostat arrays. If there is interest in this service please contact the core. 

Storage of Data:

All data files associated with a study will be placed on the BCH Secure File Transfer” system for 30 days.  After that period it will be archived for five years of free storage on the \\rc-fs\genomics-core server. However, data retrieval after the first 30 days on the secure file transfer system may be difficult and is the responsibility of the investigator. After five years, the data will be lost forever if the investigator has not retrieved and stored it elsewhere.

Storage of Samples:

Once your project is completed please pick up your samples. All samples will be discarded 90 days after completion.

Software:

The following software packages are free to use or download -
Affymetrix Command Console Software
Affymetrix Data Transfer Tool
Affymetrix Expression Console Tool
Affymetrix Genotyping Console Software

Transcriptome Analysis Console (TAC) Software
Fusion Software Developers Kit
TM4 Microarray Software Suite

Other software options for microarray analysis -

ArrayStar v2.1 from DNASTAR
GeneSifter
GeneSpring
Partek
Rosetta Resolver System

NanoString nCounter® MAX Profiler:  A simple, cost effective solution for multiplexing 10 – 800 targets in a single tube.

• Upgraded from the nCounter Sprint to the nCounter Max!
• Eliminate the need for cDNA synthesis, PCR or library prep by using this simple hybridization technology. 
• Assay DNA, RNA, miRNA or protein in 24 hours without the need for technical replicates. 
• Flexible sample input allows you to use purified RNA, cell lysate, blood, FFPE.
• Easy data analysis done at your desk with free software(nSolver software) for quick, publication quality figures.
• Pre-designed panels or custom options available through Nanostring.  For assistance, please contact Brendan Collins at <bcollins@nanostring.com>. 

We have ABI SeqStudio & SeqStudio Flex 24 capillary sequencers. Flexibility to process small and large scale projects quickly. We strive to provide a rapid turnaround time of 1-3 business days and will keep you updated about any delays.

This instrument is self service. Please request under the Equipment Reservations tab. 

Available for Self-Use. Please request under Equipment Reservations tab.

The Visium CytAssist enables the transfer of transcriptomic probes from standard glass slides to Visium slides, enabling spatial profiling insights to be gained from even more samples. Compatible with hematoxylin and eosin (H&E)- or immunofluorescence (IF)-stained tissue sections, CytAssist allows pre-sectioned tissues to be used for the Visium workflow. You can further maximize your Visium experiments by screening tissue sections using standard histological techniques to find biologically significant sections and then precisely align those sections within the Visium slide Capture Area using CytAssist.

• Simplify sample handling with facilitated transfer  of transcriptomic probes from standard slides onto the Visium Capture Area
• Expand sample compatibility to pre-sectioned  and pre-stained tissues on standard glass slides
• Maximize insights from Visium experiments by prescreening tissue sections with standard histological techniques to select biologically significant sections
• Precisely capture up to two FFPE or frozen tissue sections per run in less than one hour using  Visium CytAssist

Visium workflow with CytAssist

Facilitate transfer of transcriptomic probes in FFPE or fresh frozen samples with Visium CytAssist. In the Visium CytAssist workflow, sectioning, tissue preparation, staining (H&E or IF), and imaging take place on a standard glass slide. After probe hybridization, two standard glass slides and a Visium slide with two Capture Areas are placed in the CytAssist instrument so that the tissue sections on the standard slides can be aligned on top of the Capture Areas. Within the instrument, a brightfield image is captured to provide spatial orientation for data analysis, followed by hybridization of transcriptomic probes to the Visium slide. The remaining steps, starting with probe extension, follow the standard Visium workflow outside of the instrument.

Visium CytAssist Gene and Protein User Guides and Protocols

Space Ranger analysis pipelines

Loupe Browser visualization software

For assistance with training, please contact:

Shoshoni Droz, Ph.D.
Staff Spatial Field Application Scientist, Tissue Specialist, NE/NY/CT
shoshoni.droz@10xgenomics.com

Analysis of Bionano Saphyr data.  Price is per sample.  

Please note that the MGCF is NOT a CLIA facility.

This instrument is self service. Please request under the Equipment Reservations tab. 

The CFX384 Touch Real-Time PCR Detection System is a powerful and precise real-time PCR instrument in a 384-well format, for researchers who require both ease of use and rapid, high-throughput data acquisition. The CFX384 Touch System utilizes accurate temperature control and sensitive, 4-color detection optics to deliver reliable and repeatable qPCR results for singleplex or multiplex reactions.

Quickly set up runs and monitor amplification traces in real time on the integrated LCD touch screen, or use CFX Maestro Software to easily and intuitively design your experiment and analyze results from a connected computer. With up to four-target detection, unsurpassed thermal cycler performance, unrivaled stand-alone functionality, and powerful software, the CFX384 Touch System was designed to advance your qPCR.

Key Features and Benefits

With the CFX384 Touch Real-Time PCR Detection System you can:

• Obtain precise quantification and multiplex target discrimination – optical system uses long-lasting solid-state technology with 5 filtered LEDs and 5 filtered photodiodes
• Confirm system performance and data reliability – internal system qualification and system test software, automated data quality control, and various validation tool services are available.
• Obtain accurate results with volumes as low as 3 µl – minimize sample and reagent usage
• Use advanced data analysis tools — normalized gene expression using CFX Maestro Software
• CFX Maestro Software Works Seamlessly with PrimePCR™ Assays and Panels. The expert design and wet-lab validation of all PrimePCR Assays ensure optimal assay performance. The integration of CFX Maestro Software with PrimePCR Assays aids in the analysis and interpretation of experimental results.
• Begin analyzing data in a single step by importing target, reference, and control information from a PrimePCR run file directly into CFX Maestro Software
• Combine data from multiple plates into a Gene Study to rapidly screen large numbers of targets or samples
• Use powerful data visualization tools, such as hierarchical clustering and color-matched expression levels, to identify individual targets or clusters to consider for further investigation

Assistance with data movement and transfer.  1 hour minimum.

Price Per Two Plates

We have had to raise our prices due to a vendor raising their prices.  We are monitoring the situation and will lower our prices again as soon as we can. 

• Please send us at least 3 μg of high-quality gDNA per sample. Samples should have a concentration of at least 30-50 ng/ul in nuclease-free water or TE buffer.
• Samples should be analyzed for quality and purity by gel electrophoresis using a 1% agarose gel; must be free of RNA contamination and show no signs of degradation. (Please send us a copy of gel image along with your samples).  If you cannot do this we can do it for you.
• Samples should have an A260/A280 ≥ 1.8 and an A260/A230 ≥ 1.6.
• If both minimum ratio values listed above are not met please perform additional precipitation before shipping your samples to us. High-quality genomic DNA is absolutely essential for successful outcomes. PICOGREEN DO NOT nanodrop!!!!!! Not sufficient!

For post-PCR samples with fluorescent markers already added.

*An optimization run may be needed to find the ideal concentration. 

This service covers TCR Spectratyping and Multiplex ligation-dependent probe amplification (MLPA) plates. Please specify if you need the SeqStudio run conditions adjusted for your assay. 

Available for Self Service. Request under Equipment Reservations Tab.

LUNA-FL™ Dual Fluorescence Cell Counter by Logos Biosystems

Available for self service.  Please request from Equipment Reservations tab. 

Equipped with brightfield and dual fluorescence optics that allows the sensitive detection of most cell types excluding bacteria. The LUNA-FL™ can distinguish primary cells such as PBMCs, splenocytes, neutrophils, and stem cells from undesirable debris for accurate cell count and viability results. Brightfield cell counting, fluorescence cell counting, yeast cell counting, and GFP transfection assays are all in the LUNA-FL™ wheelhouse. 

• Four counting modes: Bright Field, Fluorescence, Yeast Cell (FDA/PI), and GFP Transfection Assay
• Customize and save protocols
• Dilution Calculator
• Gate Cells on histograms

Core provides PhotonSlide for ultra-low fluorescence counting. Each slide holds two samples.  Core does not provide stain. 

LunaFl resources and guides here.

1) Sample: The client will submit a minimum of 1.4ug of gDNA as the starting material.

2) Microarray: The microarray will be performed using Illumina’s Methylation EPIC array using related instrumentations and softwares.

3) Deliverables: Raw data in spreadsheet as the deliverable for this project.  The resulting data will be made available via FTP download.

4) TAT: Turnaround time is approximately 3-4 weeks after sample has passed initial QC.

5) Additional note: This project is for research use only.

Electronic gene mapping on the OhmX Analyzer. Contact us for details and pricing.

A member of the MGCF core will load your flow cell and monitor your run on the PromethION 24.

This services includes two wash-and-reloads, regardless of flow cell performance.

The Molecular Genetics Core Facility wil not use flow cells that do not meet warranty standards according to Oxford Nanopore Technologies. Otherwise, the Core is not responsible for variance in flow cell performance.

The MGCF will receive prepared libraries. Ligated DNA and cDNA are stable in elution buffer. Please do not add Sequencing Buffer and Library Beads before transport to the Core.

Load your own flow cell at the Molecular Genetics Core!

Full Service PromethION Ligaiton Sequencing with Nanopore reagents provided by the user.

Price is per run. 

Please book time under the Equipoment Reservations tab. 

Available for self-use for the NestSeq user group.

Contact the Core for details on joining the self-user group or requesting full service. 

Please request under the Equipment Reservations tab.

Illumina NextSeq 1000 Sequencing System offers breakthrough system design, chemistry innovations, compatibility with an expansive list of library preparation options, and onboard integrated informatics for rapid secondary analysis. A range of flow cell types and kits support a variety of customer batching and throughput needs, from bulk RNA sequencing (RNA-Seq) to 16S sequencing and shotgun metagenomics.

• Flexibility and scalability to expand the breadth of applications performed on a benchtop system
• 100-400 M reads at a variety of read lengths, including 300 bp paired-end reads
• Operational simplicity with load-and-go cartridges and integrated onboard informatics with DRAGEN
• Improved run economics with high output to support larger studies and data-intensive methods
• Lower operating costs than the MISeq and NextSeq500

Specification Sheet

Data Concordance

Oxford Nanopore Technologies' proprietary motor protein and current-enabled nanopore technology enables continuous sequencing of DNA fragments up to 2 MB and RNA fragments up to 15 kb.

Kit v14 Nanopore Ligation Chemistry enables a DNA basecalling accuracy of over 99 percent, identifies over twice as many SVs as Ilumina NGS options, and can capture DNA and RNA methylation information.

The PromethION 24 allows for high-throughput, rapid WGS of human and mouse genomes.

Rapid return of results within a week. Please reach out directly to the Core staff to schedule sample drop-off for rapid results. 

Prices change frequently - please email for updated quote.

 Molecular Genetics Core does QC and send-out.

Price is per 12 samples. 12 sample minimum charge.

*Please request under the Equipment Reservations tab and schedule a time in our calendar

User provides Qiagen kit. We provide reagent bottles, tips, tubes, and rotor adaptors necessary for the Qiacube.

The pricing is per run. A run can accomodate 2-12 samples.

Turnaround time: ~1 business day 

The Agilent 2100 Bioanalyzer system provides sizing, quantitation and quality control of DNA and RNA.

RNA Analysis

The Bioanalyzer RNA kits with the patented RNA Integrity Number (RIN)are the industry standard for RNA quality assessment, allowing reliable and precise integrity checks and sample quantitation before any RNA dependent application.

• Maximum flexibility – check total RNA, mRNA, or cRNA quality
• Minimal sample consumption – only 5 ng of total RNA or 50 pg of total RNA required for analysis
• RNA Integrity Number (RIN) – a reliable tool to compare

What the RIN can do:

• Obtain a numerical assessment of the integrity of RNA.
• Directly compare RNA samples, e.g. before and after archival, compare integrity of same tissue across different labs.
• Ensure repeatability of experiments, e.g. if RIN shows a given value and is suitable for microarray experiments, then the RIN of the same value can always be used for similar experiments given that the same organism/tissue/extraction method is used.

What the RIN cannot do:

• Tell a scientist ahead of time whether an experiment will work or not if no prior validation was done (e.g. RIN of 5 might not work for microarray experiments, but might work well for an appropriate RT-PCR experiment. Also, a RIN that might be good for a 3' amplification might not work for a 5' amplification).
• 100% guaranty a sample will amplify properly. There are some salts, phenols, and other contaminates that the Bioanalyzer cannot detect.  If these are present in a sample it may generate a good RIN but not amplify properly.

• RNA Integrity Database (RINdb) – a freely accessible repository holding hundreds of user submitted total RNA traces

Small RNA Analysis

The Agilent Small RNA kit provides a fast and sensitive analysis to resolve small nucleic acids in the size range of 6 to 150 nt.

• High resolution – miRNA smear region separated from tRNA
• Minimal sample consumption – use as little as 50 pg of purified miRNA or 10 ng of total RNA for analysis in 1 μL volume
• A faster and more sensitive alternative to agarose and polyacrylamide gels analysis for detection of small RNA
• Analyze total RNA (10-100 ng/μL), fractionated, or synthetic samples.

DNA Analysis

The DNA kits are ideal for automated sizing and quantitation of PCR fragments, restriction digests or fragmented DNA.

• Broad linear dynamic range – detection of less abundant products, e.g. non-specific amplifications
• High resolution – separate fragments down to 5 bp
• Sizing accuracy – normalization to internal markers and ladder
• Quantitation accuracy and reproducibility – quantitation of each DNA fragment against internal standards
• Minimal sample consumption – only 1 µL sample required

High Sensitivity DNA Analysis

The High Sensitivity DNA kit allows analysis of fragmented DNA or DNA libraries.

• Increased sensitivity - down to 5 pg/µL
• Analysis of complex DNA samples - down to 100 pg/µL for fragmented DNA or DNA libraries
• Broad linear dynamic range - detection of less abundant PCR products
• Smear analysis - specify size range of interest
• Quantitation accuracy and reproducibility - quantitation of each DNA fragment against internal standards
• Minimal sample consumption - only 1 µL of sample required

Free analysis software from Agilent: Agilent 2100 Expert Software

This is especially helpful with the DNA chips so you can perform the sizing to your specifcations. 

Turnaround time: ~1 business day 

The 4200 TapeStation system separates nucleic acids by electrophoresis. The system detects:

• Fluorescently stained double-stranded DNA including genomic DNA
• Fluorescently stained total RNA (Eukaryotic and Prokaryotic)

Depending on your application, the software automatically determines the size, quantity, molarity, purity, RINe, DIN, %cfDNA, or ribosomal ratios. Reports can easily be generated and saved in PDF format, or exported for editing in Microsoft Word or Excel. 

Submit a 4ul aliquot of your sample for TapeStation assays.  The sample will not be returned.  Please list concentration values in iLab form.

Customers must provide all reagents and slides. Core provides gaskets, wash buffers, and Human Cot-1.

The Agilent SureScan Scanner provides a precise and efficient microarray scanner system for many research applications. Designed for research using microarrays, the scanner system provides a laser-induced fluorescence scanning solution designed to read microarrays printed on standard 1 inch by 3 inch glass slides.

The microarray instrumentation measures the fluorescence intensity of two dyes simultaneously from labeled sample nucleic acid bound to microarrays. The system also enhances productivity with continuous loading from a 24-slide cassette and automatic loading of data files into Feature Extraction Software. It also features built-in ozone protection to minimize signal degradation.

QC analysis is provided by the core. In depth analysis can be done using the free Agilent analysis software for CGH & CGH+SNP Microarrays, CytoGenomics Software

*Please request under the Equipment Reservations tab and schedule a time in our calendar

User provides Qiagen kit. We provide reagent bottles, tips, tubes, and rotor adaptors necessary for the Qiacube.

The pricing is per run. A run can accomodate 2-12 samples.

Assistance with data movement and transfer.  1 hour minimum.

Bio-Rad's QX200 droplet digital PCR system can obtain highly precise answers about genetic makeup. Absolute quantification is achieved by partitioning 20ul samples into 20,000 nanoliter-sized droplets, which allow for thousands of discrete measurements per sample. This massive sample partitioning is a key aspect of the ddPCR technique.

Following PCR amplification, droplets from each sample are analyzed individually on the QX200 Reader, where target-positive and negative droplets are counted to provide absolute quantification in digital form. The QX200 System works with both probe and EvaGreen fluorescence chemistry.

The ddPCR system can be used to:

• Detect rare DNA target copies (e.g. somatic mutations with low VAF)
• Determine copy number variation and gene expression with unmatched accuracy
• Precisely detect genome editing events
• Provide an absolute, standard-free measure of library concentration and quality, optimizing NGS performance overall

Available for full or self service. Contact Julia Phoon with questions/training requests: Julia.Phoon@childrens.harvard.edu

Helpful Links:

Planning Droplet Digital PCR Experiments

Droplet Digital PCR Applications Guide

Droplet Digital PCR Assay Finder or

PrimePCR Search Tools

QuantaSoft Analysis Pro Software- free analysis software

If you are unsure about what services you need or have a complex project, please click on the "contact the Core" tab at the top of the page and send us a message asking for clarification or a consultation.

Customers must provide all reagents and IFCs. Prices are subject to change depending on usage.

Regardless of chip format (48.48, 96.96, 192.24), samples should be submitted as follows:

TaqMan RNA Expression: At least 10ul of each cDNA sample (w/o preamp or 1:5 dilution of preamped) and 10ul of each assay (20X) in separate V-shape 96-well plates.

EvaGreen RNA Expression: At least 10ul of each cDNA sample (w/o preamp or 1:5 dilution of preamped) and 10ul of each primer pair (20uM) in separate V-shape 96-well plates.

When replicating samples or assays, please adjust the submission volume accordingly.

Data Deliverable

Your data will be available to download when a request is completed. Please go to "View My Requests" tab, click blue toggle icon to expand the request, and go to Attachments & URLs section, where you can find one or two zipped files (per chip), depending on the detection chemistry you choose. 

You will need the Fluidigm Real-Time PCR Software to analyze data for each service. User guides are also provided below. Please note that the software is designed to run on Windows machines only. For Mac users, please notify the core staff.

Real-Time PCR Analysis

Real-Time PCR Analysis User Guide

We will check sample quantity and quality for all full-service projects before we begin amplification.

Agilent Bioanalyzer

 We use the Agilent Bioanalyzer to check sample quality before starting amplification. The Bioanalyzer software automatically assigns an RNA Integrity Number (RIN) to a eukaryote total RNA sample. Using this tool, sample integrity is no longer determined by the ratio of the ribosomal bands, but by the entire electrophoresis trace of the RNA sample. This includes the presence or absence of degradation products. The assigned RIN is independent of sample concentration, instrument, and analyst, therefore becoming a de facto standard for RNA integrity.

RNA Integrity Number (RIN) – a reliable tool to compare samples

What the RIN can do:

• Obtain a numerical assessment of the integrity of RNA.
• Directly compare RNA samples, e.g. before and after archival, compare integrity of same tissue across different labs.
• Ensure repeatability of experiments, e.g. if RIN shows a given value and is suitable for microarray experiments, then the RIN of the same value can always be used for similar experiments given that the same organism/tissue/extraction method is used.

What the RIN cannot do:

• Tell a scientist ahead of time whether an experiment will work or not if no prior validation was done (e.g. RIN of 5 might not work for microarray experiments, but might work well for an appropriate RT-PCR experiment. Also, a RIN that might be good for a 3' amplification might not work for a 5' amplification).
• 100% guarantee that a sample will amplify properly. There are some salts, phenols, and other contaminants that the Bioanalyzer cannot detect.  If these are present in a sample it may generate a good RIN but not amplify properly.

 RNA Integrity Database (RINdb) – a freely accessible repository holding hundreds of user-submitted total RNA traces

Nanodrop

 We use the Nanodrop to check sample concentration and purity providing the sample is not below the detection limit of 2ng/ul. Using the 260/280 and 260/230 ratios we can help confirm sample purity.

• The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol, or other contaminants that absorb strongly at or near 280 nm.
• The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants that absorb at 230 nm, such as EDTA, carbohydrates, phenol, TRIzol, guanidine HCL, and guanidine isothiocyanate.

Full Service:

Samples should be prepared according to the following instructions and delivered to the Core Facility at CLS 16030.23. All samples for Affymetrix should be submitted in 1.5 mL centrifuge tubes labeled with the sample name or number including the following recommended amounts.

GeneChips for high quality/high concentration RNA – 100ng to 500ng of total RNA in 5ul, plus 2ul of total RNA (around 50ng/ul) for bioanalyzer QC.
GeneChips for lower quality(FFPE)/low concentration – 100pg to 10ng of total RNA in 5ul, plus 2ul of total RNA (around 50ng/ul) for bioanalyzer QC.

Submitting more than the required amount of samples is ok as we will normalize all your samples after we perform the QC.

GeneChip miRNA Arrays-

 Hyb-only Service:

GeneChip 3′ Expression  – 15ug biotinylated cRNA in less than 32ul.
GeneChip Exon, ST,HTA, Clariom Arrays – 10ug  of ssDNA( fragmented and terminal labeled) in 60ul.
GeneChip miRNA Arrays- 21.5ul biotin labeled sample

We are currently not running the CytoScan, OncoScan, or Karyostat arrays. If there is interest in this service please contact the core. 

Storage of Data:

All data files associated with a study will be placed on the BCH Secure File Transfer” system for 30 days.  After that period it will be archived for five years of free storage on the \\rc-fs\genomics-core server. However, data retrieval after the first 30 days on the secure file transfer system may be difficult and is the responsibility of the investigator. After five years, the data will be lost forever if the investigator has not retrieved and stored it elsewhere.

Storage of Samples:

Once your project is completed please pick up your samples. All samples will be discarded 90 days after completion.

Software:

The following software packages are free to use or download -
Affymetrix Command Console Software
Affymetrix Data Transfer Tool
Affymetrix Expression Console Tool
Affymetrix Genotyping Console Software

Transcriptome Analysis Console (TAC) Software
Fusion Software Developers Kit
TM4 Microarray Software Suite

Other software options for microarray analysis -

ArrayStar v2.1 from DNASTAR
GeneSifter
GeneSpring
Partek
Rosetta Resolver System

NanoString nCounter® MAX Profiler:  A simple, cost effective solution for multiplexing 10 – 800 targets in a single tube.

• Upgraded from the nCounter Sprint to the nCounter Max!
• Eliminate the need for cDNA synthesis, PCR or library prep by using this simple hybridization technology. 
• Assay DNA, RNA, miRNA or protein in 24 hours without the need for technical replicates. 
• Flexible sample input allows you to use purified RNA, cell lysate, blood, FFPE.
• Easy data analysis done at your desk with free software(nSolver software) for quick, publication quality figures.
• Pre-designed panels or custom options available through Nanostring.  For assistance, please contact Brendan Collins at <bcollins@nanostring.com>. 

Price is per run. 

Please book time under the Equipoment Reservations tab. 

Analysis of Bionano Saphyr data.  Price is per sample.  

Please note that the MGCF is NOT a CLIA facility.

Electronic gene mapping on the OhmX Analyzer. Contact us for details and pricing.

For post-PCR samples with fluorescent markers already added.

*An optimization run may be needed to find the ideal concentration. 

This service covers TCR Spectratyping and Multiplex ligation-dependent probe amplification (MLPA) plates. Please specify if you need the SeqStudio run conditions adjusted for your assay. 

This instrument is self service. Please request under the Equipment Reservations tab. 

We have had to raise our prices due to a vendor raising their prices.  We are monitoring the situation and will lower our prices again as soon as we can. 

• Please send us at least 3 μg of high-quality gDNA per sample. Samples should have a concentration of at least 30-50 ng/ul in nuclease-free water or TE buffer.
• Samples should be analyzed for quality and purity by gel electrophoresis using a 1% agarose gel; must be free of RNA contamination and show no signs of degradation. (Please send us a copy of gel image along with your samples).  If you cannot do this we can do it for you.
• Samples should have an A260/A280 ≥ 1.8 and an A260/A230 ≥ 1.6.
• If both minimum ratio values listed above are not met please perform additional precipitation before shipping your samples to us. High-quality genomic DNA is absolutely essential for successful outcomes. PICOGREEN DO NOT nanodrop!!!!!! Not sufficient!

1) Sample: The client will submit a minimum of 1.4ug of gDNA as the starting material.

2) Microarray: The microarray will be performed using Illumina’s Methylation EPIC array using related instrumentations and softwares.

3) Deliverables: Raw data in spreadsheet as the deliverable for this project.  The resulting data will be made available via FTP download.

4) TAT: Turnaround time is approximately 3-4 weeks after sample has passed initial QC.

5) Additional note: This project is for research use only.

Available for self-use for the NestSeq user group.

Contact the Core for details on joining the self-user group or requesting full service. 

Please request under the Equipment Reservations tab.

Illumina NextSeq 1000 Sequencing System offers breakthrough system design, chemistry innovations, compatibility with an expansive list of library preparation options, and onboard integrated informatics for rapid secondary analysis. A range of flow cell types and kits support a variety of customer batching and throughput needs, from bulk RNA sequencing (RNA-Seq) to 16S sequencing and shotgun metagenomics.

• Flexibility and scalability to expand the breadth of applications performed on a benchtop system
• 100-400 M reads at a variety of read lengths, including 300 bp paired-end reads
• Operational simplicity with load-and-go cartridges and integrated onboard informatics with DRAGEN
• Improved run economics with high output to support larger studies and data-intensive methods
• Lower operating costs than the MISeq and NextSeq500

Specification Sheet

Data Concordance

Prices change frequently - please email for updated quote.

Price Per Two Plates

Available for Self Service. Request under Equipment Reservations Tab.

LUNA-FL™ Dual Fluorescence Cell Counter by Logos Biosystems

Available for self service.  Please request from Equipment Reservations tab. 

Equipped with brightfield and dual fluorescence optics that allows the sensitive detection of most cell types excluding bacteria. The LUNA-FL™ can distinguish primary cells such as PBMCs, splenocytes, neutrophils, and stem cells from undesirable debris for accurate cell count and viability results. Brightfield cell counting, fluorescence cell counting, yeast cell counting, and GFP transfection assays are all in the LUNA-FL™ wheelhouse. 

• Four counting modes: Bright Field, Fluorescence, Yeast Cell (FDA/PI), and GFP Transfection Assay
• Customize and save protocols
• Dilution Calculator
• Gate Cells on histograms

Core provides PhotonSlide for ultra-low fluorescence counting. Each slide holds two samples.  Core does not provide stain. 

LunaFl resources and guides here.

 Molecular Genetics Core does QC and send-out.

Price is per 12 samples. 12 sample minimum charge.

If you are unsure about what services you need or have a complex project, please click on the "contact the Core" tab at the top of the page and send us a message asking for clarification or a consultation.

Customers must provide all reagents and IFCs. Prices are subject to change depending on usage.

Regardless of chip format(48.48 or 96.96) you choose, samples should be submitted as follows:

TaqMan Genotyping: At least 10ul of each human gDNA sample at 60ng/ul* and 10ul of each assay (80X) in separate V-shape 96-well plates.

SNPtype Genotyping: At least 10ul of each human gDNA sample at 60ng/ul* and 10ul at 100 uM of each LSP, STA primer, and ASP1/ASP2 primer in separate V-shape 96-well plates.

 * The required concentration depends on the genome size of species.

When replicating samples or assays, please adjust the submission volume accordingly.

Data Deliverable

Your data will be available to download when a request is completed. Please go to "View My requests" tab, click blue toggle icon to expand the request, and go to Attachments & URLs section, where you can find one or two zipped files(per chip), depending on the detection chemistry you choose.

You will need Fluidigm Genotyping Software to analyze data from each service. User guides are also provided below. Please note that the software is design to run on windows machine only. For Mac users, please notify the Core staff. Exported excel file will be provided instead. If assistance is needed, please contact fluidigm@iddrc.org.

Genotyping Analysis

Genotyping Analysis User Guide

We have ABI SeqStudio & SeqStudio Flex 24 capillary sequencers. Flexibility to process small and large scale projects quickly. We strive to provide a rapid turnaround time of 1-3 business days and will keep you updated about any delays.

Available for Self-Use. Please request under Equipment Reservations tab.

The Visium CytAssist enables the transfer of transcriptomic probes from standard glass slides to Visium slides, enabling spatial profiling insights to be gained from even more samples. Compatible with hematoxylin and eosin (H&E)- or immunofluorescence (IF)-stained tissue sections, CytAssist allows pre-sectioned tissues to be used for the Visium workflow. You can further maximize your Visium experiments by screening tissue sections using standard histological techniques to find biologically significant sections and then precisely align those sections within the Visium slide Capture Area using CytAssist.

• Simplify sample handling with facilitated transfer  of transcriptomic probes from standard slides onto the Visium Capture Area
• Expand sample compatibility to pre-sectioned  and pre-stained tissues on standard glass slides
• Maximize insights from Visium experiments by prescreening tissue sections with standard histological techniques to select biologically significant sections
• Precisely capture up to two FFPE or frozen tissue sections per run in less than one hour using  Visium CytAssist

Visium workflow with CytAssist

Facilitate transfer of transcriptomic probes in FFPE or fresh frozen samples with Visium CytAssist. In the Visium CytAssist workflow, sectioning, tissue preparation, staining (H&E or IF), and imaging take place on a standard glass slide. After probe hybridization, two standard glass slides and a Visium slide with two Capture Areas are placed in the CytAssist instrument so that the tissue sections on the standard slides can be aligned on top of the Capture Areas. Within the instrument, a brightfield image is captured to provide spatial orientation for data analysis, followed by hybridization of transcriptomic probes to the Visium slide. The remaining steps, starting with probe extension, follow the standard Visium workflow outside of the instrument.

Visium CytAssist Gene and Protein User Guides and Protocols

Space Ranger analysis pipelines

Loupe Browser visualization software

For assistance with training, please contact:

Shoshoni Droz, Ph.D.
Staff Spatial Field Application Scientist, Tissue Specialist, NE/NY/CT
shoshoni.droz@10xgenomics.com

A member of the MGCF core will load your flow cell and monitor your run on the PromethION 24.

This services includes two wash-and-reloads, regardless of flow cell performance.

The Molecular Genetics Core Facility wil not use flow cells that do not meet warranty standards according to Oxford Nanopore Technologies. Otherwise, the Core is not responsible for variance in flow cell performance.

The MGCF will receive prepared libraries. Ligated DNA and cDNA are stable in elution buffer. Please do not add Sequencing Buffer and Library Beads before transport to the Core.

Load your own flow cell at the Molecular Genetics Core!

Full Service PromethION Ligaiton Sequencing with Nanopore reagents provided by the user.

Oxford Nanopore Technologies' proprietary motor protein and current-enabled nanopore technology enables continuous sequencing of DNA fragments up to 2 MB and RNA fragments up to 15 kb.

Kit v14 Nanopore Ligation Chemistry enables a DNA basecalling accuracy of over 99 percent, identifies over twice as many SVs as Ilumina NGS options, and can capture DNA and RNA methylation information.

The PromethION 24 allows for high-throughput, rapid WGS of human and mouse genomes.

Rapid return of results within a week. Please reach out directly to the Core staff to schedule sample drop-off for rapid results. 

This instrument is self service. Please request under the Equipment Reservations tab. 

The CFX384 Touch Real-Time PCR Detection System is a powerful and precise real-time PCR instrument in a 384-well format, for researchers who require both ease of use and rapid, high-throughput data acquisition. The CFX384 Touch System utilizes accurate temperature control and sensitive, 4-color detection optics to deliver reliable and repeatable qPCR results for singleplex or multiplex reactions.

Quickly set up runs and monitor amplification traces in real time on the integrated LCD touch screen, or use CFX Maestro Software to easily and intuitively design your experiment and analyze results from a connected computer. With up to four-target detection, unsurpassed thermal cycler performance, unrivaled stand-alone functionality, and powerful software, the CFX384 Touch System was designed to advance your qPCR.

Key Features and Benefits

With the CFX384 Touch Real-Time PCR Detection System you can:

• Obtain precise quantification and multiplex target discrimination – optical system uses long-lasting solid-state technology with 5 filtered LEDs and 5 filtered photodiodes
• Confirm system performance and data reliability – internal system qualification and system test software, automated data quality control, and various validation tool services are available.
• Obtain accurate results with volumes as low as 3 µl – minimize sample and reagent usage
• Use advanced data analysis tools — normalized gene expression using CFX Maestro Software
• CFX Maestro Software Works Seamlessly with PrimePCR™ Assays and Panels. The expert design and wet-lab validation of all PrimePCR Assays ensure optimal assay performance. The integration of CFX Maestro Software with PrimePCR Assays aids in the analysis and interpretation of experimental results.
• Begin analyzing data in a single step by importing target, reference, and control information from a PrimePCR run file directly into CFX Maestro Software
• Combine data from multiple plates into a Gene Study to rapidly screen large numbers of targets or samples
• Use powerful data visualization tools, such as hierarchical clustering and color-matched expression levels, to identify individual targets or clusters to consider for further investigation