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Agilent: Overview & FAQ
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The PMGC is one of only a handful of Agilent Certified Microarray Service Providers, and was the first to receive this designation in Canada. To maintain this designation, our technicians participate in a rigorous certification program. This ensures that the customer is provided with the best possible service. The PMGC makes use of Agilent’s SurePrint mciroarrays. This inkjet driven manufacturing process offers a great deal of flexibility and ensures that Agilent is able to maintain content that is frequently updated to the newest builds of the various genomes.

                                                  


Pricing: Please inquire
Pricing is highly dependent on experimental design - please allow us to help you find the most cost effective solution, take advantage of our complimentary experimental design consultation!

Contact:

Agilent Service Coordinator: Monika Sharma


Quick Questions:

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What does it mean to be a “Certified Service Provider”?

Agilent selects a small number of service labs with the ability to follow a rigorous certification process. This involves additional on-site training as well as an assessment of technical capability while following Agilent's robust protocols. For the PMGC, this process was completed for all 4 platforms (Gene Expression, ChIP-on-Chip, aCGH, and miRNA). Click here to learn more about what it means to be an Agilent Certified Service Provider.

Where can I find more information about Agilent array content?

The PMGC has developed a powerful tool (ArrayTrans) to allow for researchers to easily search for genes or probes represented on Agilent human and mouse arrays. For additional information about the content of Agilent microarrays, including species other than human and mouse, please visit Agilent’s eArray tool.

How much total RNA (or DNA) is required for an Agilent microarray?

The amount of material required depends on the application. For gene expression experiments 150-300 ng of total RNA is required. For miRNA experiments we require 100 ng of total RNA. For array CGH experiments we require require 3 µg of gDNA. Please refer to the specific pages for Gene Expression, miRNA or aCGH profiling for more accurate guidelines on sample requirements.

Is there an amplification kit available for small samples (nanogram range)?

The Low RNA Input Fluorescent Linear Amplification Kit can handle samples sizes as low as 50 ng. Please contact us () for more details.

What are my options if I have degraded RNA such as that obtained from FFPE extractions?

We do not currently offer a solution for profiling FFPE RNA on Agilent arrays. For degraded RNA we recommend either the Affymetrix GeneChips® coupled with the NuGen Ovation FFPE WTA system or Illumina arrays using DASL technology.

Is the performance of the arrays impacted by the RNA isolation technique/kit used?

We have not seen a major impact on the performance of the arrays using well-established RNA extraction methods, so long as the quality of the RNA is comparable. One important metric is the 260/230 ratio which should be over 1.8. Some extraction methods that use organic reagents can result in low 260/230 ratios due to carry over of the reagents, and this can impact labelling. It is important however that you use the same extraction method for all samples within one project.

What are the advantages and disadvantages between 1-colour and 2-colour experimental design?

The 2-colour design was originally developed to reduce the error rate associated with variability in microarray manufacturing. As the technology has matured and protocols have become much more robust, the need for 2-colour designs was largely negated. Agilent has continued to offer 2-colour functionality for customers who prefer this design based on a legacy from the original microarrays produced. The major advantage of a 1-colour design is in cost reduction, which can often be of significance to a researcher.

Are there controls spotted on the Agilent arrays?

Each Agilent array contains a number of positive and negative controls. There are two main types of negative probes – those that have been designed structurally to form hairpins (i.e., the probe folds back upon itself and self-hybridizes) and thus should not hybridize to any labelled sample. The second is a probe designed to not be complementary with any species and thus should give a minimal signal (effectively zero). For positive controls Agilent uses a combination of spike in probes some created internally and some from the ERCC (External Reference RNA Consortium).

We have created a custom array with Agilent. Can your service perform experiments using them for us?

Absolutely. We will require you to provide us with the arrays or with collaborator status on eArray. The cost of the service will be adjusted if the arrays are not included in the pricing. We will require the design file (.xml) from eArray in order to quantify the arrays. All other terms of service remain the same.

What steps are taken to ensure the least amount of variability?

Microarray experiments are relatively complex, with numerous steps, reagents and instruments involved. Despite this, it has been shown in many studies that the leading causes of variance are both the facility that is performing the analysis followed by the technician that completes the experiment. All of our technicians are highly experienced and rigorously trained. Each project is assigned a specific technician who completes the entire project to minimize variability. Furthermore, wherever possible, we use arrays and reagents from the same lot. We use the same equipment (hybridization oven, scanner, etc.) throughout the project; all in an effort to ensure the tightest possible data.

Often a project is large enough that it will require multiple days worth of amplification, labelling, and hybridizing to complete. In these cases we work with the customer to identify which samples are replicates and we split these replicates across the various days to ensure that it is possible to control for any day-to-day variations that may occur.

What is the difference between a technical replicate and a biological replicate? Which type is most useful and how many replicates do I need?

A biological replicate involves independent samples (multiple patients, multiple biopsies from an individual patient, etc.). RNA or DNA would be extracted from unique biopsies, blood from unique patients or from independent cell cultures (i.e. individual culture dishes). The purpose of a biological replicate is to assess and control for biological diversity.

A technical replicate involves splitting a sample at some point and continuing on with the two aliquots as separate samples through the rest of the protocol. So for example, a technical replicate might involve taking one RNA sample and performing two independent amplifications and labellings from that initial sample. Similarly, if a labelled sample was split onto two arrays, that would be another type of technical replicate. Technical replicates provide an indication of measurement (or technical) error and are useful for diagnosing problems with the protocol, but offer little in the way of statistical power for a biological experiment.

The exact number of replicates required for an experiment is difficult to determine a priori without a proper power analysis. Such a power analysis is not always possible as it requires that you have an estimation of the overall variance, which you often do not have before you perform the experiment. We generally recommend doing as many biological replicates as your budget can accommodate. In general it is good to have at least three biological replicates per condition. For a more detailed determination of the number of replicates required, please contact us as we will be happy to help you design your experiment.

What kind of data will I receive with the optional data analysis?

All customers running an Agilent experiment through the PMGC will receive image files (both ".tiff" and ".jpg" formats) and raw data files from the Feature Extraction software (as ".txt", ".xml", ".csv" and ".shp" files).

Customers who opt for our additional data analysis service will also receive files from a quality control assessment, completed in R, as well as the appropriate statistics, heat-map and fold-change analysis for their data set. For a more detailed answer specific to the particulars of your experiment, please inquire with Carl Virtanen, our Bioinformatics Manager, at .