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Microarray gene expression experiments allow the expression of thousands (even hundreds of thousands) of genes to be profiled simultaneously. These experiments are often performed to monitor the changes in gene expression caused by a certain treatment, disease state, or particular stage in development, thus enabling researchers to gain insight into the mechanistic actions of novel therapeutics, the progression of disease states, and developmental stages. Gene expression profiling can be performed on the Affymetrix, Agilent and Illuminaarray platforms.

Recently, great strides have been made in RNA amplification technologies thus allowing clinical samples, laser-capture microdissected cells, FFPE samples, and even single cells to be profiled. Various RNA amplification techniques, including methods developed by Affymetrix, Agilent, Illumina, Miltenyi Biotec, NuGEN, Sigma, and Dr. Norman Iscove, have been used at the UHNMAC. Please contact us to discuss the most appropriate amplification method for your experiment.

Gene expression experiments can be either 1- or 2-colour designs. 1-colour experiments (available on Affymetrix, Agilent and Illumina platforms) allows for changes to the experimental design of a project (suitable for projects that take place over a long period of time with an unknown number of samples), while 2-colour experiments (available on Agilent platforms) make more direct comparisons between two samples but require a more rigid experimental design. Please contact us to discuss your experimental design.

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Quick questions:
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How many replicate experiments should I do?

We recommend doing a minimum of three replicates. For UHNMAC Services, three replicates are required if data analysis is to be performed. Three replicates is the minimum number of replicates required before analysis can be performed with even minimal confidence.

What is the difference between a technical replicate and a biological replicate? Which type is best?

A biological replicate involves isolating RNA independently from replicate sources (multiple patients, multiple biopsies from an individual patient, etc). The purpose of a biological replicate is to control for biological diversity. Biological replicates are often more telling, and for this reason are “better” than technical replicates, however, biological replicates are often more difficult to obtain.

A technical replicate would be a multiple labeling or reciprocal labelling of the same RNA sample. This replicate may be useful in some cases (when one slide has poor signal for example) but it has little true statistical value when the experiment works.

I only have enough RNA to do two experiments. Should I request a reciprocal labelling?

With the Indirect Labelling method (where amino-allyl dUTP is incorporated and the dye is conjugated after the reverse transcription reaction), we have found that reciprocal labelling is not necessary, especially when there is only enough sample for two experiments. Reciprocal labelling is recommended when directly labelling cDNA as the dyes are incorporated with different efficiencies. This is the reason we highly recommend indirect labelling for all two-colour experiments.

How much total RNA is required for labelling?

The amount of RNA varies depending on the platform of microarray. In general We require a minimum 200 ng of total RNA at a minimum concentration of 100 ng/μL, per standard labelling reaction. As a standard quality check procedure, we run each sample on the Agilent Bioanalyzer prior to labelling. So additional total RNA will be required. Please contact us for details.

For substandard or degraded samples, such as those extracted from FFPE tissues or laser captured microdissected samples, or samples that have low amount of input RNA, such as those from sorted cells, RNA amplification can also be performed. Please contact us for details.

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

In microarray experiments, one of the largest sources of variability is the technician performing them. All of our technicians are experienced, highly trained individuals and, whenever possible, the same technician will perform all experiments in a project. Whenever possible, we use arrays from the same print batch, reagents from the same lot, and use the same equipment (hyb oven, scanners, etc) to reduce array-to-array variability