FAQs - Microarray Experimental Design
Array Design
We are available to assist you in project design, cDNA library synthesis, and experimental design. Variables that can be modified to suit your research include:
Type of Array
Most custom microarrays presently used to study expression levels are comprised of PCR amplified cDNA. These arrays have the benefit of utilizing cDNA libraries without the necessity of sequence verification and have the flexibility to be fabricated for organisms with little to no genomic documentation. Gene specific primers can also be used to amplify genes from a library if sequences are available. The disadvantage to this type of arrays is that genes with lower expression levels and those with longer transcripts are often more difficult to obtain in a library and may not be represented on an array without extensive normalization techniques such as size fractionation and subtractive hybridization. Front end labor is also relatively high with cDNA arrays
Oligonucleotide arrays offer elements with normalized Tm’s and concentrations to decrease variability of intensities and hybridization kinetics across the array. Disadvantages of oligo arrays are that gene sequences must be known and probes have to be designed with high specificity. Oligo size can vary from 50-80bp and can be purchased in 96-well format from several companies.
Number of Elements on Array
Size of the array is dependent on the size of the project. For analysis purposes, larger arrays often minimize deviation during normalization; however, preparing more elements to be spotted can be costly. A simple way to get around this is to print replicate spots or replicate arrays on a single slide. Replicate supergrids, are cost affective ways to replicate data points and minimize effects of background fluorescence without having to perform additional hybridization experiments. Currently, our Chipwriter Pro instrument can generate 30,000 features on an array. This allows for 15,000 spots in duplicate supergrids.
Control spots and normalization techniques
There are currently three popular techniques to normalize for dye intensity biases:
Global normalization: This method scales mean intensities of every spot found on the array to a set number for each channel. The scaling ratio for that given channel can then be applied to individual intensities to give a normalized intensity. This technique requires no control spots, but only works well with medium to large arrays (>5000). The idea in using this form of normalization is that individual genes may be up regulated or down-regulated in a given experiment; however, the differences in overall expression levels must be accounted for. As one can imagine, this is not the ideal technique for some experiments that lower total expression such as extreme cold. This should also not be applied to arrays consisting of solely possible genes of interest.
Control genes: The second normalization method relies on the use of “housekeeping genes”, or genes that are responsible for basic functions found in every cell and experience little to no expression differences. Unless proven within your experimental parameters to have no differentiation in expression levels, this may not be a reliable technique with which to normalize your data.
Spike controls: The third method requires the addition of control spots and RNA spikes into your reaction. A control DNA is usually chosen from an organism in an entirely different kingdom than that being studied. Often, plant or bacterial genes are used in animal arrays and vice-versa. Equal amounts of RNA spikes for that particular control gene are added to each experimental sample and normalization can be applied to the mean of these spot intensities over each channel. This technique mainly corrects for technical variations in the sample preparation protocol.
Purification for Printing
Spot morphology is an extremely important aspect of array manufacturing and data analysis. Ideal microarrays will consist of uniformly circular features of DNA spotted equidistantly on the array surface. Although the idea sounds simple with the use of the proper robotics, several variables contribute to hydrophobicity and volatility of the printing material. Salt concentration plays a very important role in spot size and therefore needs to be made uniform from sample to sample. We offer 96-well PCR purification with the use of the Biomek FX robotic workstation. DNA is purified by ultrafiltration to remove buffer salts, nucleotides and primers from the PCR product.
An aliquot of the sample can be set aside and sequenced on our ABI3700 in the GATC Sequencing Facility. The remaining material will be lyophilized until the arrays are ready for fabrication.
Array Fabrication
After the printing material is purified and resuspended in printing buffer, the Bio-Rad Chipwriter Pro is used to fabricate the arrays. This microarray printer relies on Telechem Stealth split pin technology to deliver equally sized spots with low variability of DNA concentration. The flexibility of the software allows virtually any format of array and can manufacture up to 100 slides and a maximum of 48 pins at once. Currently, glass slides are bought through the core facility at the rate extended to us by the vendor. Users may also supply their own printing substrates if they wish.
After slides are printed, they are stored at room temperature in under desiccation until needed for hybridization. Our online data management system keeps track of inventory of your personal arrays and informs you when more slides need to be fabricated.
