Massively Parallel Sequencing Facility
ABOU T U S
Personnel
Manager:
Tim Hunter
Senior Research Technician:
Scott Tighe
Rates
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Project Consultation Free
RNA Assessment Nano, Pico, Small RNA, DNA 1-12 Samples/$30
Target Prep (Std. affumetrix) $175
Nugen Ovation. Target Prep $200
Nugen Pico Ovation Target Prep $200
Double Spia Amplification $25
Target Prep for Exon or Gene Arrays $225
Test Chip (including Test Chip) $175
Target Hyp/Scan (User provides target chip) $75
Turnaround Time
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Project Consultation By appointment
RNA Assessment Nano, Pico, Small RNA, DNA 24-48 hrs
Target Prep (Std. affumetrix) 5-10 busines days
Nugen Ovation. Target Prep 5-10 business days
Nugen Pico Ovation Target Prep 5-10 business days
Target Prep for Exon or Gene Arrays 5-10 business days
Test Chip (including Test Chip) 5-10 business days
Target Hyp/Scan (User provides target chip) 2-3 business days
Important Forms
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RNA Quality Assessment Order Form (DOC)
RNA Submission Form: Target Preparation (DOC)
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Sample Quality Control and Results Form (QC for Staff Only) (PDF)
Contact Us
Health Science Research Facility
149 Beaumont Avenue, Room 305
Burlington, VT 05405
Phone: (802) 656-2557
Official Website
The UVM Microarray Core Facility was initiated by the Vermont Genetics Network and
established in 2002 in collaboration with the Vermont Cancer Center and UVM College of
Medicine. The facility utilizes the Affymetrix GeneChip system for microarray analysis, the
NanoDrop spectrophotometer and Qubit for critical nucleic acid quantification, the Agilent 2100
Bioanalyzer for DNA and RNA analysis, and the MP Biomedicals Fast Prep-24 for extracting
RNA or DNA from difficult tissues or sample types. Services offered include: RNA extraction
from difficult sample types, training for proper RNA handling and extraction, global gene
expression profiling for eukaryotes and prokaryotes using the standard 3' arrays, gene and exon
arrays for human and mouse, DNA mapping (SNP) studies, and custom arrays. The facility staff
has developed protocols for novel sample types including formalin fixed paraffin embedded
tissues for DNA mapping studies, high recovery isolation of nucleic acids from LCM and FACS
sorted samples, and prokaryotic target preparation from limited quantity RNA samples. All
microarray projects are reviewed using an integrated approach to experimental design, workflow,
and sample collection in collaboration with the UVM Bioinformatics Core Facility to ensure
high quality, statistically relevant results. The facility also sponsors user educational forums such
as technology seminars, tutorials, general lectures, workshops, and an annual open house.
Affymetrix GeneChip technology provides efficient access to genetic information using
miniaturized, high-density arrays of oligonucleotide probes. The arrays are manufactured by
Affymetrix's proprietary, light-directed chemical synthesis process, which combines solid-phase
chemical synthesis with photolithographic fabrication techniques employed in the semiconductor
industry. We encourage you to check our website prior to starting any new experiment. This will
provide you with useful information and may answer some of your questions.
The Core is not responsible for lost or damaged chips left in our possession after your
experiment is completed. If the Gene Chip array is found to be faulty at the time of hybridization
and/or analysis, a new array will be provided by Affymetrix at no cost, but the core facility
charges will still apply. It can take 4 weeks or more for Affymetrix to send a replacement chip.
Data are made available to you in the form of a DVD. We suggest that you make your own
backups. The Core is not responsible for lost data.
RNA samples exhibiting a large degree of degradation will not be recommended for downstream
processing. Please note that, if you ask us to process such a sample we will NOT be responsible
for poor results. A second analysis will NOT be done free of charge.
NEVER provide the facility with ALL your stock RNA.
It is recommended to retain a small volume for future RNA studies and can be amplified if
needed to pursue more studies with this sample.
Users assure that all RNA samples were obtained with appropriate animal research committee
approval and human subjects IRB approval. Any samples obtained from cells infected with
potential pathogens must be declared before submission.
The UVM Microarray Core Facility reserves the right to use all data from hybridizations
performed in the facility for the purpose of assessing quality of data generated in the facility and
for the development of quantitation and analysis tools. The data will be used in a strictly
anonymous.
Services
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Experimental design consultation (jointly with Bioinformatics Group)
mRNA (transcriptome) sequencing
Methylation sequencing
miRNA (ncRNA) sequencing
ChIP sequencing
Metagenomics
Amplicon sequencing
de novo sequencing
Exome sequencing
Targeted capture sequencing
Discounted pricing on many consumables and reagents through core facility negotiations
Equipment
Affymetrix GeneChip Platform including
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7G Scanner
(2) FS-450 Fluidics Stations
Agilent 2100 Bioanalyzer
NanoDrop Spectrophotometer
Qubit Spectrophotometer
MP Biochemicals FastPrep24
BioSpec Mini Bead Beater
Techne TC-312
Bio-Rad DNA engine with 96 well block and dual block alpha units
Eppendorf microcentrifuge 5415C
Eppendirf microcentrifuge 5415D
Computer Workstation
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Dell Optiplex 755
Dell Optiplex 330
Software loaded includes
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XRAY Biotique
GeneSifter
GCOS (Gene Chip Operating System)
CNAT (Copy Number Analysis Tool)
GTYPE (Genotype calls)
Expression Console
Submission
Guidelines
Step 1: Initial Consultation. All investigators who wish to pursue a project through the facility
are strongly encouraged to meet with the Microarray and Bioinformatics Facility staff together to
ensure proper experimental design and a clear understanding of sample requirements for
processing through the facility including turnaround times. All projects requesting bioinformatic
support are required to meet with the bioinformatic staff to draft a design file. Please contact Tim
Hunter at 656-2557 or Jeff Bond at 656-4068 to arrange a consultation meeting.
Step 2: Submit Total RNA samples for quality assessment. All RNA samples must be DNase
treated prior to submission to ensure all contaminating genomic DNA has been removed. Please
submit an RNA quality Assessment Order Form to the intake area of the facility (samples should
be placed in the freezer in 305A). It is recommended that you isolate your RNA using TriZol
followed by Qiagen's RNeasy kit; although, we have found that in most cases RNeasy works
well for cell culture and TriZol for tissue(protocols can be picked up in the facility or
downloaded from the website. Information required on the form include RNA concentration
(ng/ul), 260/280 ratio, RNA source, and isolation method. Concentration is critical since the
dynamic range of the 2100 Bioanalyzer is 10-500 ng/ul for the NanoChip and 200pg/ul-10ng/ul
for the PicoChip. We require 2.5 ul of clean RNA for sample assessment. We recommend that
you resuspend or elute your RNA in DEP-C water. The facility staff will meet to discuss results
and a report including traces will be given back to the investigator and consulted how to proceed.
Once you have submitted your samples for Bioanalyzer chip analysis, you are considered to be in
the queue for target preparation.
Step 3: Submit RNA samples for Target prep. A Target Prep Form will need to be submitted
in the intake area (HSRF 305). Information required is RNA concentration, and A260/280 ratio.
Eukaryotic RNA: The concentration of total RNA [not mRNA] MUST be =20ng/ul. 40-50ng of
total RNA is recommended for each sample, but the amount can be modified due to system
constraints [see below under limited RNA availability].
Prokaryotic RNA: The required amount of RNA is 10 ug or greater at a concentration NO LESS
THAN 500 ng/ul. Alternative methods have allowed the use of 4.5ug but only for special
requests. Generally, 4.5-10 ug have generated very good data. For total RNA quantities less than
4 ug, please discuss with the staff. For LCM prokaryotic samples [i.e 100ng], it is possible to
synthesize Poly -A tials and amplify with the Nugen Ovation system. This is experimental only
and employing this technique will only be performed after serious discussion with the core staff.
**Limited RNA availability: For eukaryotic samples at 200pg to 10ng/ul or total amounts of 110 ng, an alternative sample preparation protocol can be used in the microarray facility. The
Nugen Pico RNA amplification system has performed very well on LCM samples and other
manually micro-dissected tissues. Any RNA at these concentrations is eligible. For prokaryotic
samples at less than 250 ng/ul [4ug total], please discuss this with the microarray staff.
FACS Sorting for RNA: Please consult with the microarray staff for successful RNA recovery
from cells sorted using flow cytometry. Several key points are highlighted below:
Preparing the flow cytometry is not a small task as it must be completely free of RNases from the
sheath tank to the sorting nozzle. This decontamination procedure will take considerable time, so
be prepared. Ensure the dip tubes, septa, flow cell, all tubing lines, and nozzles have been
completely decontaminated with bleach, RNase ZAP, ethanol, autoclaving, or other qualifying
technique prior to the sort. Rinse cytometer with DEPC water. Remember DEPC water DOES
NOT inactivate RNases, and is only RNase-free water. The sheath fluid and tank must be RNase
free. Use only sterile RNase free tubes on the cytometry that have never been open to
contaminated air.
As a control, retain some cells and extract the RNA to determine the condition of RNA prior to
the sort. Also before and after trypsinization for adherent cells. Often the flow cytometry, even
after a good decontamination procedure can be the source of RNases. Whenever possible
suspend cells in RNAlater prior to the sort and after trypsinization. Again, checking RNA
integrity at this step is a good control. If RNAlater is not an option, you may consider adding
RNase inhibitor to prevent degradation during a sort. Use RNase inhibitors that are DTT
independent such as Ambion's Superase-In.
During the sort, sort cell directly into your extraction reagent, something like Trizol LS or
Qiagen's RLT. When using TriZol, Only Use TriZol LS which is slightly more concentrated than
regular regular TriZol. This formula allows lower quantities of reagent to be used relative to the
amount of the sample. (Regular TriZol can tolerate 10 part TriZol to1 part sample volume while
TriZol LS can allows 3:1. In the case of Guanidium Isothiocyanate (or Qiagens's RLT buffer)
sort at a ratio of 100ul of sort liquid to 350 ul of RLT or a multiple of that.
Immediately after the sort, extract RNA according to the selected reagents manufacturer's
protocol and evaluate the integrity of the RNA.
Step 4: Test Chip Hybridization and Scanning. This is optional. The test chip can assess the
efficiency of the cDNA reaction and quality of the target prep intensities before moving on to the
more expensive target array. An experiment report will be provided to the investigator.
Step 5: Hybridization and scanning of target chip. A DVD will be provided to the investigator
including the .dat, .cel, and .chp files. Absolute text files or excel files can be generated upon
request. The facility will archive a copy, but we suggest that you also make your own back-up.
The facility will generate a report summarizing all steps, quality control checks, and results for
potential troubleshooting.
Step 6: Data Analysis. Detailed analysis is the responsibility of the investigator in collaboration
with the Bioinformatics Core.
Sample Submission, Storage, and Retrieval Policy
Qualitative Assessment using the Bioanalyzer
Samples brought to the microarray facility for total RNA, mRNA, cDNA, or DNA quality
assessment should be in a rack or box labelled with PI name and placed in the -20 freezer labeled
"Microarray" located in 305 HSRF. DO NOT BRING YOUR STOCKS. Please bring a 2ul
aliquot in a 0.5 ml. microcentrifuge tube that is labeled on the top of the tube and corresponds
exactly with what is written on the order form. Make sure RNA or DNA is at the proper
concentration range for the chip analysis requested. All unused aliquots will be tossed away after
the assessment is completed and the data uploaded to your BioDesktop account or emailed.
Microarray Target Preparation: RNA or DNA
Samples for target preps for new expression profiling projects should be submitted as 200ng total
RNA at a concentration of 20ng/ul or greater (for lower concentrations see the Facility staff).
Samples for ongoing expression studies using the standard Affymetrix target prep should be
submitted as 3 ug of total RNA at a concentration >250ng/ul. Genomic DNA for mapping
studies should be submitted as 400ng total DNA at a concentration of 40ng/ul or greater. It is
critical that this DNA be resuspended in T10E0.1 (see staff if you need some of this reagent).
Regardless of sample type, place samples with tops labeled in rack or box with PI name in the
Microarray -20 freezer located in 305 HSRF. Make sure appropriate order forms are submitted
and sample names are matched exactly between the order form and labeled tube. DO NOT
SUBMIT YOUR STOCKS. RNA or DNA left over from target preps will be stored for six
months and will be tossed by the facility staff at their discretion without prior notification.