General

Our labeling and hybridization protocols are optimized for MWG microarrays.  We currently use PerkinElmer (PE) Cy3/Cy5 chemistry for labeling which means that each RNA sample (control and experimental) can be labeled with a different dye, combined and hybridized to the same slide.   Each dye signal is then detected on a PE ScanArray Express Scanner.  PE ScanArray Express software allows for quantitation and comparison of the Cy3 and Cy5 signals.  Your microarray data will be sent on a CD that will include raw data, TIFF images for Cy3 and Cy5 jpg file with a Cy3/Cy5 combined image, and a basic Quantarray analysis.   We recommend that you become familiar with GeneSpring software for microarray data analysis.  GeneSpring allows for in-depth and user-defined analysis of your data.  We will provide training in the use GeneSpring software on request.

 Experimental Design 

We perform standard and flip labeling for all experiments.  Flip labeling (“dye swap”) eliminates dye bias associated with unequal incorporation of the two Cy dyes into cDNA.  Standard and flip labeling should be done with RNA isolated at different times, from different set of cells or from fresh tissue (biological repeat), rather than with the same RNA (technical repeat).

There are two types of basic experimental design: reference design and balanced block design.  The major difference between these two is that reference design requires twice as many slides than balanced block design.  However, balanced block design can only be used only for simple class comparison type of analysis (looking for genes that are upregulated or downregulated between two samples).  Reference design is far superior for more complex class comparison as well as for class prediction or class discovery where clustering analysis is required.  In fact, clustering analysis can not be performed on experiment conducted using balanced block design.  As a rule reference design should be used when amount of material is a limiting factor, and balanced block design should be used when number of slides is limited. 

Reference RNA for reference design experiments used does not necessarily need to be physiologically relevant to your RNA.  However, reference design should have at least 80% coverage for the particular slide (i.e. should contain mRNA representing at least 80% of genes on the slide).  RNA isolated from a cells or tissues usually has 50-60% coverage.  Reference RNA is, most often, a pool of RNA from several different cell lines or tissues.  Human, mouse, and rat universal reference RNAs are available from several vendors (Stratagene, Promega…..)

 http://www.stratagene.com/products/showProduct.aspx?pid=439)

There is relatively high False Detection Rate (FDR) associated with microarray experiments.  With 5% confidence, one can expect 1,000 false positive genes from high density microarray of 20,000 spots.  Therefore it is important to repeat microarray experiments.  We currently recommend repeating microarray experiment 3 times (one microarray experiment consisting of two slides – standard and flip, with total of 6 biological repeats).  For RNA isolated from tissues additional repeat(s) may be needed.  Slide price, amount of starting material, quality of data are some of the factors that should be considered when decision about number of repeats is made. The following web site from Richard Simon at the National Cancer Institute includes manuscripts and talks about microarray experimental design and number of repeats:
http://linus.nci.nih.gov/~brb/TechReport.htm.

 RNA Quantity and Quality

 The quality of your RNA is the single most important factor in determining the outcome of your microarray analysis.  The total RNA that you submit should be intact (not degraded) and free of protein and DNA contamination.  The quality of every RNA sample will be quality tested by measuring the A260/A280 ratio and by nanochip analysis on the Agilent Bioanalyzer.  Agilent has developed software that will assign a specific quality number to the RNA sample based on its electrophoretic profile.  The RNA Integrity Number (RIN) ranges from 1 (totally degraded RNA) to 10 (completely intact RNA).  Only high quality RNA, with RIN greater than 7 and A_260/280 greater than 1.8 will be considered for microarray analysis.  Your total RNA samples will also be quantified using Nanodrop spectrophotometry.

 Good laboratory practice and RNase-free solutions must be used during total RNA isolation.  Most commercial total RNA isolation kits are acceptable since they produce high quality RNA.  Column based kits eliminate the possibility of genomic DNA contamination.  However, yield with these kits is usually lower than with kits that don’t use columns (e.g. Trizol, and TRI reagent).  If you are using Trizol, TRI reagent or other phenol containing solution for RNA isolation, do not carry over any DNA from the water/phenol interface.

 Isolation of RNA from viable cells usually gives intact high quality RNA.  However isolation of RNA from tissues tends to be problematic.  If you are isolating RNA from tissues, effort should be made to isolate RNA as soon as possible (or at least tissue frozen in liquid nitrogen).  Ambion markets a product called RNAlater © which allows the investigator to preserve tissues for RNA isolation at a later time. 

 Dissolving the purified RNA pellet can be difficult.   An effective protocol for dissolving RNA pellet is as follows.  After final wash in 70% or 75% alcohol, spin down your RNA for 10 min at 4⁰C, and pipette out the solution.  Repeat this until there is no more solution left.  Air dry pellet for 1 min and dissolve it in a small volume of RNase-free water (20-40 ul) by pipetting up and down until pellet is completely dissolved.  (Do not allow the RNA pellet to dry out for long periods because it is almost impossible to dissolve RNA pellet.)  Vortex solution for 1 min and then incubate at 55⁰C for 10 min.  Then vortex again for 1 minute and spin briefly.  Since we use an Invitrogen direct labeling kit from that requires 10-40 mg of total RNA, we prefer to receive a minimum of 20 mg total RNA per labeling reaction.  RNA should be dissolved in RNase-free water and final concentration should be > 1.5 mg/ml.

 Microarray Slides

MWG microarrays are made by spotting 50-mer oligonucleotides onto epoxy treated glass slides.  MWG has a wide variety of arrays and for the most current list you can visit MWG web site at http://ecom2.mwgdna.com/services/array/download.tcl.  You may download the complete list of genes on each MWG slide. If you want to know which arrays contain your gene(s) of interest, you can access Compact Gene Index Search option on the same page.  The Compact Gene Index can be accessed only from European site and it is necessary to register with MWG to use this service. 

 Microarray slides are relatively expensive ranging in price from $200 to $1,000.   We stock commonly used MWG mouse, human and rat slides.  If you require special slides, please notify us at least two weeks in advance so that the slides can be received in a timely manner.  

Turn-around Time 

Given the time required for the completion of RNA analysis (one day), microarray labeling and hybridization (two days) and initial data analysis (2-3 days), the turn-around time will be at least 5 days from the time you submit your RNA samples, assuming that there is not a backlog of other requests.

 Results of microarray experiment should be confirmed by another RNA quantification technique such as real-time PCR, Northern blot, or RNase protection assay.  ABI and Bio-Rad real time thermal cyclers are available on a sign-up basis.  Applied Biosystems (ABI) offers real-time PCR kits for many genes (“assays on demand”)
 http://myscience.appliedbiosystems.com/navigation/mysciApplications.jsp?tabNameAttribute=applGeneEx.

 If you have any questions about the service or pricing, please contact:

Goran Boskovic, Ph. D.
Microarray Core Facility Manager

E-mail: gboskovi@marshall.edu

Department of Microbiology, Immunology and Molecular Genetics
Marshall University
Joan C. Edwards School of Medicine
1542 Spring Valley Drive
Huntington, WV 25704
Phone: (304) 696-7271
Fax:      (304) 696-7354