UTK Affymetrix Core Facility Sample Submission Detail
1) You must contact the core facility director to discuss experimental design and
sample preparation prior to submitting any samples for a project.
(affycore@utk.edu or 865-974-5244). Replicates (n=3) are HIGHLY
SUGGESTED if you want sound data. Most peer reviewed journals will now
only accept data sets that are replicated.
2) Total RNA extracts are all that is needed. Spec readings should be in the
range of 1.8-2.1 in TE and 1.6-1.9 in water.
3) 1-10 µg of RNA per sample is needed for the standard protocol. The
minimum concentration must be 0.5 µg/µl.
4) 100ng of RNA is needed for the two cycle or amplified RNA protocol.
5) When samples are ready, call the core to ensure arrays are in house.
6) The account will be charged for arrays that are ordered for an
anticipated project, but not used, if another user cannot be found before
the expiration date (approximately one year).
7) The primary investigator must sign the submission form and give a valid
account number (UTK) or P.O. number (ORNL, ETSU, UTK Med. Hospital).
An invoice will be sent or the funds transferred (UTK only) once the arrays
have been successfully processed. Procurement cards cannot be used.
Both arrays and processing will be billed as supply recovery. Arrays range in
price from $100-$400. Processing for each array is $400 (to cover reagents,
personnel time and instrument maintenance fees).
8) Records should be maintained for each sample according to MIAME
(Minimum Information about a Microarray Experiment) Standards see
www.mged.org/miame for details. Please include on the submission form the
sample name, experiment name, group or replicate number, tissue type and
which samples should be used for baseline comparison (if any). The
Affymetrix Core will maintain records on sample preparation, hybridization
and scanning procedures.
9) Each sample will be run on the Agilent 2100 Bioanalyzer to check for
degradation. The investigator will be contacted if samples do not pass this
initial control check.
10) Samples will be spectrophotometrically checked after synthesis of cDNA and
IVT reactions. If these reactions fail, the PI will be contacted.
11) The arrays will be QC checked following scanning according to the protocols
of Affymetrix. Which can be downloaded at the following address:
www.affymetrix.com/support/technical/manual/expression_manual.affx
QC Criteria are 1) inspection of image, 2) B2 Oligo Performance and grid
alignment, 3) Average background and noise values, 4) internal and spikein controls, 5) percent present, 6) scaling and normalization factors.
12) The data analysis output (.chp file) will be saved as a .txt file which can be
loaded into Excel as a tab delimited file. The file will contain the raw signal,
detection and detection p-value for all probe sets.
13) Current turn around time ranges from 1 week to 4 weeks. Arrays are
processed in order the samples are received!
14) The Affymetrix Core will help with data analysis if needed. We currently use
Stratagene (Iobion) ArrayAssist and Pathway Assist or Decision Site’s
SpotFire for analysis. These analysis packages are approximately $1500 for
a one seat annual license. UTK is also developing a Stanford Microarray
Database Analysis site for containing freeware at UTK. The Affymetrix Core
has a workstation computer with these programs loaded and users are
welcome to sign up for access time. The Core will provide each user with an
introductory session (approximately 1 hour) on how to use the software
packages. Online Data Tutorials are also provided for each software package
and users are urged to learn how to use the programs themselves. The Core
is also in the process of obtaining dedicated analysis personnel. Please note:
Data analysis of microarrays can be a very tedious process. You may have
~40,000 data points per array. If your grant proposal is due next week, do not
anticipate having your data sufficiently analyzed in time for submission!
15) Send total RNA overnight on dry ice to the following address:
UTK Affymetrix Core Facility
Attn: Julia Gouffon
1215 W. Cumberland Ave.
229 Jessie Harris Building
Knoxville, TN 37996