Roche NimbleGen | Gene Expression

Advantages

Our extensive catalog of gene expression array designs contains the major eukaryotic model organisms and over two hundred prokaryotic organisms. In addition to our standard 385K array format, Roche NimbleGen offers two multiplex formats, 4x72K and 12x135K, to ensure complete genome coverage of both small prokaryotic and large eukaryotic genomes without compromising information content or data quality. You can choose to process and hybridize your samples in your lab using our optimized protocols and reagents. Alternatively, send your samples to us for processing in our full-service laboratory, results guaranteed.

Multiplex Options: A cost-effective solution for high sample throughput

NimbleGen multiplex arrays enable you to analyze statistically meaningful populations for alterations in expression levels of annotated genes. We offer a 4-plex format (4x72K) with 72,000 probes per array and a 12-plex format (12x135K) with 135,000 probes per array. Hybridize four or twelve independent samples or run replicate samples on a single slide and average the data for increased statistical confidence.

Up-To-Date Microarray Designs

Roche NimbleGen's digital microarray manufacturing process is fast and inexpensive, allowing you to get any sequenced genome on an array rapidly. All designs are built from the latest published genomes to ensure your experiment is current.

High Information Content: Multiple probes per transcript enhance confidence in results

Ultra-high density NimbleGen arrays (385,000 and 2.1 million probes) allow every gene to be represented by multiple, unique probes. The averaging of the signal from multiple probes provides improved statistical confidence, reducing the impact of inconsistent probe behavior and improving the signal-to-noise ratio compared to platforms that offer fewer probes per gene.

Long Oligos: Long oligonucleotide probes enhance the accuracy of your analysis

NimbleGen has an unmatched ability to synthesize high-density arrays of long oligos. For gene expression analysis, long oligos (60mer) provide superior signal-to-noise ratio, increased sensitivity, specificity, and discrimination, particularly in complex eukaryotic genomes. Another significant advantage: long probes eliminate the need for mismatch probes, effectively doubling array capacity.

Great Inter-Array Reproducibility

NimbleGen arrays are manufactured to exacting standards and undergo rigorous quality control to ensure superior array-to-array data reproducibility. In replicate hybridization experiments, inter-array r² values routinely exceed 0.98.

Expression Analysis Software

ArrayStar® software from DNASTAR, Inc. enables scientists and researchers to open and analyze normalized data (.call, .calls and .txt files) generated from Roche NimbleGen’s Expression arrays.

NimbleGen Array Formats

Format Options

Roche NimbleGen offers three array formats for our Gene Expression products:

385K - Our industry standard 385,000 probe format provides gene expression arrays with highest information content on the market
4x72K - Four individual samples may be hybridized and analyzed simultaneously on each array with 72,000 probes.
12x135K - Running on our recently launched HD2 platform, 12x135K offers 12 arrays per slide with 135,00 probes per array.

Array Specifications

	385K	4x72K	NEW! 12x135K^*
Number of Arrays per Slide	1	4	12
Total Number of Probes	385,000	4 x 72,000	12 x 135,000
Probe Length	60mer	60mer	60mer
Number of Probes per Target^**	6 - 20	2 - 7	3 - 12
Number of Replicate Probe Sets per Array^**	1 - 4	1 - 3	1 - 2
Feature Size	16 x 16μm	16 x 16μm	13 x 13μm
Array Size	17.4 x 13mm	7.8 x 5.7mm	8.9 x 6.5mm
Slide Size	1 x 3 in. (25 x 76 mm)
^* Available for delivery only.
^** Depending on transcriptome size.

NimbleGen Array Workflows

Workflow Options

Roche NimbleGen offers two workflow options for our Gene Expression arrays:

Delivery - You hybridize your samples and analyze the data in your lab using our optimized protocols and reagents. Available for both catalog and custom designs on all array formats.
Service - You send your samples to us for processing in our full-service laboratory; results guaranteed. Currently available for 385K and 4x72K array formats only.

Workflow Availibility

Delivery and Service workflows are not available for all Gene Expression designs and formats. Use the table below to identify which workflows are currently available:

	385K Format	4x72K Format	12x135K Format
Eukaryotic Designs	Delivery and Service	Delivery and Service	Delivery
Prokaryotic Designs	Delivery and Service	Delivery and Service	N/A
Custom Designs^*	Delivery	Delivery	Delivery
^* Contact your local sales representative for service option availability.

Service Workflow Overview

Roche NimbleGen's gene expression microarray service consists of the following steps:

Customer selects catalog array design, or works with Roche NimbleGen bioinformatics scientists to design a custom array.

Customer supplies RNA or cDNA. To ensure sufficient sample for quality control analysis as well as optimum hybridization, we require the following amounts of total RNA, mRNA, or cDNA:

	Total RNA*	mRNA*	cDNA
Eukaryote	20μg	5μg	2.5μg
Class I Prokaryotes	20μg	-	2.5μg
Class II and III Prokaryotes	30μg	-	5μg
*Please note that the NimbleGen procedure for sample processing does not include RNA amplification.

Roche NimbleGen performs the expression hybridization, scans the array, extracts the data, and performs a preliminary data analysis.
Roche NimbleGen delivers the following data and software to customer:
- Microarray Design Files
- Probe Sequences
- Gene Descriptions
- Raw Data Files
- Normalized Data Files

Delivery Workflow Overview

Customers can purchase catalog arrays or custom arrays from NimbleGen and perform the array experiments at their own laboratories or core facilities. NimbleGen arrays are synthesized on standard-sized glass microscope slides, compatible with many microarray scanners. NimbleGen offers a line of hybridization instruments, kits and consumables as well as provides a complete user’s guide to support customers with sample labeling, array hybridization, scanning, data extraction, and gene analysis. Please contact Roche NimbleGen for a list of required equipment and reagents. NimbleGen also offers a training program to get you up and running with NimbleGen arrays quickly.

Software

Literature

NimbleGen Gene Expression Microarrays and Services
Brochure (PDF Format 1MB)
NimbleGen Eukaryotic Gene Expression HD2 12-Plex Array Delivery
Datasheet (PDF Format 365KB)
NimbleGen Prokaryotic Gene Expression 4x72K Microarrays
Datasheet (PDF Format 480KB)
NimbleGen Zebrafish Gene Expression 385K Arrays
Datasheet (PDF Format 361KB)
NimbleGen Arrays User’s Guide: Gene Expression Analysis
User’s Guide (PDF Format 2.9MB)
High-Quality Data Obtained with NimbleGen Gene Expression Arrays
Technical Note (PDF Format 125KB)
An Overview of NimbleGen Expression Scientific Publications
Datasheet (PDF Format 74KB)

For a complete listing of literature covering all Roche NimbleGen products and services please visit our literature page.

FAQ

Multiplex

What is the advantage of the NimbleGen 4-plex (4x72K) array format?	The NimbleGen 4x72K array format allows for the simultaneous hybridization of 4 samples on a single slide. You can conveniently run either replicates of the same sample or multiple samples on the same slide, thus reducing cost.
How many features are on each array of the NimbleGen 4x72K format?	Each array on the NimbleGen 4x72K slide has 72,000 features.
Do you offer multiple probes per transcript in the NimbleGen 4x72K format?	Yes, each transcript is interrogated by 3 or more probes, depending on the complexity of the organism. Please see our catalog for more details for each organism.
What kind of controls do you include on each array to ensure accurate sample loading and to confirm the integrity of the experiment?	Each array contains probes for Sample Tracking Controls (STCs) that are used for the following purposes: a) To identify the samples after hybridization; each labeled cDNA sample is spiked with a unique Sample Tracking control before loading. b)To ensure that there is no sample mix up between arrays.
Do all arrays on a NimbleGen 4x72K slide have the same design?	Yes. Currently all arrays on a gene expression multiplex array have identical designs.

Array Design

Do you include microRNAs or non-coding RNA in your catalog designs for expression?	Currently, no microRNA or non-coding RNA regions are used for NimbleGen gene expression array designs.
What is the probe length in gene expression arrays?	NimbleGen expression arrays contain 60-mer probes.
Do you offer catalog and custom array designs?	Yes, we offer both catalog and custom array designs for differential expression analysis. Please check our website for the current catalog. If you do not see your organism, please contact the Account Manager for your region to discuss a custom design.
Are your gene expression probes designed with a 3' bias?	Yes, probes are designed with some 3' bias. Our expression arrays are designed by first determining the transcript terminator as annotated in NCBI. We then take 1500bp upstream of that location and design our probes within that region.
Which strand is represented on the array?	The sense strand is represented on our arrays.
Can NimbleGen design a custom expression tiling array for my specific research needs?	Yes, NimbleGen can design and deliver to you custom expression tiling microarrays with probe spacing and probe length that best fits your experimental goals. Please contact the Account Manager for your region to discuss a custom design.
Are NimbleGen designs up-to-date with the latest genome builds?	Yes, NimbleGen technology allows the rapid production of new array designs based on the latest genome builds.
Are designs based on earlier genome builds available?	Yes, please contact the Account Manager for your region.

Sample Preparation

What is the recommended protocol for isolating RNA?

We recommend the RNeasy Mini Kit (Qiagen, Cat. No. 74104) or the TRIzol Reagent (Invitrogen, Cat. No. 15596-026)). If using TRIzol-isolated RNA for cDNA synthesis, it is beneficial to perform a secondary cleanup step using RNeasy Mini Kit. Immediately after the ethanol precipitation step in the TRIzol procedure, proceed with the RNeasy Mini Kit according to the manufacturers recommendations.

Can I use a different RNA isolation method than recommended?

Any RNA isolation method that provides RNA which meets the purity and integrity criteria outlined by NimbleGen can be used.

How do you assess the quality of RNA and cDNA samples?

Pure and intact RNA and cDNA should have A260/A280 and A260/A230 ratios of at least 1.8. Additionally, they should appear intact when analyzed by gel electrophoresis or using a Bioanalyzer (Agilent).

How much RNA or cDNA do you need for gene expression analysis?

This depends on whether you are sending the samples to NimbleGen for full service or if you are doing the experiment in your lab, and whether your sample is from eukaryotes or prokaryotes. When performing experiments in your lab using NimbleGen-recommended protocol, 10 μg total RNA, 1 μg mRNA, or 1 μg cDNA is required. For service, the amounts required are listed in the table below.

	Total RNA*	mRNA*	cDNA
Eukaryote	20μg	5μg	2.5μg
Class I Prokaryote	20μg	-	2.5μg
Class II and III Prokaryotes	30μg	-	5μg
*Please note that the NimbleGen procedure for sample processing does not include RNA amplification.

What is the expected cDNA yield from 10μg total eukaryotic RNA?

Using our standard protocol, we routinely get at least 5μg cDNA from 10μg total RNA.

If I do not have 10μg of RNA, what method for RNA amplification can I use in my own lab?

We have successfully used the MessageAmp II aRNA Amplification kit (Ambion, Cat. No. 1751) for RNA amplification of two human reference samples: Universal Human Reference RNA (Stratagene, Cat. No. 740000) and Human Brain Reference RNA (Ambion, Cat. No. AM6050). As with any amplification, there is a risk of introducing bias into the experiment.

What kind of samples are recommended for hybridizing on NimbleGen gene expression arrays?

We recommend using Cy3-labeled cDNA sample on our gene expression arrays. Please contact the Account Manager for your region to obtain a recommended protocol for preparing Cy3-labeled cDNA.

Data Analysis

What method of normalization does NimbleGen use for gene expression?	We normalize expression data using quantile normalization. See Bolstad B, et al. (2003), A comparison of normalization methods for high density oligonucleotide array data based on bias and variance. Bioinformatics, 19:185-193.
How does NimbleGen generate gene calls from gene expression arrays?	Gene calls are generated using the Robust Multichip Average (RMA) algorithm. See Irizarry R. et al. (2003), Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4:249-264.
What software do you recommend for further analysis of gene expression data?	NimbleGen provides gene expression values in calls file that can be conveniently imported into multiple gene expression analysis software such as ArrayStar (DNAStar). Currently, with the purchase of a full service project, you will obtain a 30-day demo version of ArrayStar along with your data. Array delivery customers can also request this 30-day demo version from NimbleGen.
Can NimbleGen gene expression data be used with publicly available software packages?	Several analysis packages that have been used with NimbleGen gene expression data are: 1. NMPP: Described in Bioinformatics (2006); 22: 2955 - 2957 2. MeV: Described in J. Exp. Biol. (2007); 210: 1507 - 1517. 3. R: Available at www.bioconductor.org, requires data in XYS format.

Array Service

How much RNA or cDNA do you need for gene expression analysis?

	Total RNA*	mRNA*	cDNA
Eukaryote	20μg	5μg	2.5μg
Class I Prokaryote	20μg	-	2.5μg
Class II and III Prokaryotes	30μg	-	5μg
*Please note that the NimbleGen procedure for sample processing does not include RNA amplification.

When I use NimbleGen full service option, what deliverables will I receive ?

NimbleGen delivers the following data files and software to full service customer:
- Microarray Design file containing probe sequences (ndf)
- Gene Description file (ngd)
- Raw Data files (calls and pair)
- Normalized Data files (RMA_calls and RMA_pair)
- ArrayStar v2.1 expression analysis software.

Array Delivery

Can I purchase NimbleGen arrays for use in my own lab or core facility?	Yes, all catalog and custom arrays are available for delivery.
Which gene expression designs are available for array delivery?	All catalog designs currently listed on NimbleGen website and any custom design are available for delivery.
What kits are available for use with NimbleGen microarrays?	NimbleGen offers Hybridization kits to facilitate efficient and specific hybridization to our expression arrays. See our Kits & Consumables web page for more details about these kits.
What resolution scanner do I need for scanning NimbleGen arrays?	A scanner with 5 μm resolution is required.
Can I scan NimbleGen arrays using my Agilent scanner?	The 5 micron Agilent scanner can work with NimbleGen arrays.
What software do I need to extract my data from a scanned image?	Roche NimbleGen’s NimbleScan software can be used to do analysis and visualization of data generated from Gene Expression arrays.
Does NimbleGen offer training for array delivery customers?	Yes, NimbleGen offers training at your lab or at our Madison, WI location. Please see our Training webpage for more details and a current schedule.
When I order microarrays from NimbleGen, what deliverables will I receive?	For microarray orders, NimbleGen delivers the following items to the customer: - NimbleGen arrays - Complete User Guide - Microarray Design file containing probe sequences (ndf) - Gene Description file (ngd) - ArrayStar expression analysis software (upon request)
How much sample do I need to run one microarray analysis in my lab?	When using NimbleGen-recommended protocol for cDNA synthesis and labeling, 10 μg total RNA, 1 μg mRNA, or 1 μg cDNA is required for each array.