NimbleGen Expression Analysis

Lab Introduction and Consultation

NimbleGen expression array product line includes a large selection of cataloged, isothermal oligo arrays covering many different organisms. Custom array designs are also possible. All expression arrays are designed for single-channel transcriptome analysis and, unlike NimbleGen's tiling array platform, these arrays are intended for single-use. For an overview of the NimbleGen microarray platform and product line, please visit their website.

The Genomics resource has three 4-plex Maui Hybridization Systems and two GenePix 4000B scanners that allow for complete processing of NimbleGen array products.

The following are instructions on how Fred Hutchinson and other Cancer Consortium members can access NimbleGen microarray services at FHCRC. Those new to this technology or new to using the Genomics Resource are strongly encourage to meet with lab personnel prior to planning a project. For non-consortium members interested in services please contact the Genomic Resource at (206) 667-2714.

Contacts:

• For questions regarding experimental design issues, please contact Jeff Delrow at jdelrow@fhcrc.org or (206) 667-2763.



• For questions regarding service fees, scheduling, and protocols please contact Andy Marty at jamarty@fhcrc.org or (206) 667-1393.

Purchasing NimbleGen Arrays

Each investigator is required to purchase arrays directly from NimbleGen. A list of current array pricing obtained from the sales contact listed below.

Roche NimbleGen Sales Contact:
David L. Helmreich
Sr. Account Manager, Northwest
Office: (503) 524-4380
Mobile: (503) 330-4363
Email: dhelmreich@NimbleGen.com

Service option 1: Preparation of Total RNA for Expression Analysis

Total RNA sample requirements are at least 4ug of total RNA at a concentration no less than 0.2ug/ul. While the lab will perform QC analysis of all samples submitted for microarray analysis, it is advisable that the investigator perform a preliminary assessment of sample quality before sending samples to the lab. Failure to perform standard QC analysis of your RNA samples will result in sample processing delays.

The Genomics Resource staff generates Cy3-labeled ds-cDNA targets for hybridization with strict adherence to sample preparation protocols outlined in the Ambion Message Amplification protocols and NimbleChip Arrays User's Guide for NimbleGen Gene Expression Analysis.

Samples should be delivered to the DNA Array Lab, located in the Thomas Building, DE-740, accompanied with a fully completed sample submission form.

NimbleGen request form:
FHCRC, Cancer Consortium members, and External users please use the sample submission form here.

Service option 2: Preparation of user supplied ds-cDNA for Expression Analysis

Double strand cDNA sample requirements are at least 6ug of ds-cDNA at a concentration of no less than 0.4ug/ul. While the lab will perform QC analysis of all samples submitted for microarray analysis, it is advisable that the investigator perform a preliminary assessment of sample quality before sending samples to the lab. Failure to perform standard QC analysis of your RNA samples will result in sample processing delays.

The Genomics Resource staff generates Cy3-labeled ds-cDNA targets for hybridization with strict adherence to sample preparation protocols outlined in the FHCRC-NimbleGen labeling protocol.

Samples should be delivered to the DNA Array Lab, located in the Thomas Building, DE-740, accompanied with a fully completed sample submission form.

NimbleGen request form:
FHCRC, Cancer Consortium members, and External users please use the sample submission form here.

Service option 3: Submitting Cy3 Labeled ds-cDNA for Expression Analysis

Researchers should submit their samples as a dried pellet with the appropriate quantities as outlined below.

Cy3-labeled cDNA	Array Type	Mixer	Quantity
Prokaryotic	1-Plex	SL	6.5ug
Eukaryotic	1-Plex	SL	13ug
Prokaryotic	4-Plex	X4	2ug
Eukaryotic	4-Plex	X4	4ug

Please provide accurate quantities as this is critical to the success of your hybridization. See the NimbleGen Gene Expression Users Guide

Samples should be delivered to the DNA Array Lab, located in the Thomas Building, DE-740, accompanied with a fully completed sample submission form.

NimbleGen request form:
FHCRC, Cancer Consortium members, and External users please use the sample submission form here.

Service options 1-3: Sample QC

Quality control of total RNA includes:
QC test for RNA degradation. The resource has two Agilent 2100 Bioanalyzers that are used to assess the initial RNA quality. To learn more about how to interpret Bioanalyzer results, please visit the Agilent web site.

QC test for protein and organic contaminates. The resource has a NanoDrop ND-1000 spectrophotometer that is used to assess sample purity. Employing a UV scan from 210nm - 310 nm, the following criteria must be met:

• 1.8 ≤ A₂₆₀/ A₂₈₀ ≤ 2.1
• A₂₆₀ / A₂₃₀ ≥ 1.7
• A₃₁₀ ~ 0

To ensure that the above criteria are based on sufficient absorption measurements, we also require an A₂₆₀ > 0.3 when performing this analysis.

In instances where one or more samples do not pass our initial QC tests, we will not proceed with the project and immediately contact the submitter with our findings.

Process Target Sample on NimbleGen Array

Adhering to FHCRC Genomics Resource SOPs and NimbleGen protocols, the Genomics Resource Lab will add additional hybridization buffers when necessary and hybridize the labeled targets overnight to the array type requested. Following hybridization, the array undergoes post-hybridization washes as dictated by FHCRC Genomics Resource SOPs and NimbleGen protocols.

Imaging of Processed NimbleGen Array

Once the array has been processed, it is scanned by the Genomics Resource Lab to generate a TIFF image file. The lab is equipped with two GenePix 4000B scanners, which are used to acquire post hybridization microarray images. The software used to control the GenePix 4000B scanners (GenePix Pro 6.0) can also be used to visualize and extract quantitative data from saved microarray images in the format of TIFF files.

Place Files on Server for Researcher Access

Data generated within the resource are transferred through automated pipelines to systems supported by the Hutchinson Center's Research Computing Support Shared Resource. Data is transferred to the user's 'fred' account, in the researcher's dnaarray folder.

If you do not currently have an account on the FRED server please fill out the Data Storage - FRED Account User Account Application prior to the completion of your microarray experiment.

Further Processing\Analysis

For more information on downstream data normalization please see the DNAStar web page.

Additionally, there are many third party software applications that can be used to further process and analyze your microarray data. Some of the software applications available to you can be found on the Genomics IT web page. (License agreements dictate that some of the software is available only from FHCRC based computers. If you are not at FHCRC and need access to the software, please contact Genomics and we will arrange access for you.)

Additional support is available to those requiring assistance with analyzing and interpreting their data. Please contact one of the following for guidance:

Jeff Delrow, Ph.D.
Director and Staff Scientist, Genomics Resource
voice : 206.667.2763
email : jdelrow@fhcrc.org

Martin Morgan, Ph.D.
Director and Staff Scientist, Bioinformatics Shared Resource
voice: 208.667-2793
email: mtmorgan@fhcrc.org