Contents€¦ · bridisation data due to the signi cantly higher signals compared to DNA...

G u i d e l i n e s f o r M y A r r a y S e r v i c e

Development of an experimentally validated microarray designfor gene expression analysis

Version 4, October 2016

For further information, please contact:

Email: [email protected]: +49 33 02 20 71 395

Contents

1 Introduction 2

2 Recommendations for Samples 4

3 Sample Shipping 6

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MyArray Service, October 2016 www.oak-labs.com

1 Introduction

Microarrays are available for a variety of different species. However, for most of the se-quenced organisms cost-efficient gene expression analysis is hampered by the nonavailabilityof microarrays. Our MyArray service is changing just that. In a two-step strategy we developa microarray design that contains only experimentally validated oligonucleotides for eachgene. These arrays allow for a cost-efficient and high-quality gene expression analysis.

MyArray Procedure

Figure 1 illustrates the steps of our MyArray service. In brief:

� An XL-design is developed based on the sequence data that is provided by the customeror publicly available. 10-20 different oligonucleotides are generated for each gene.

� An RNA sample that is comprised of RNAs from different tissues or treatment (complexRNA sample) is hybridised to one XL-microarray.

� A genomic DNA sample is hybridised to a second XL-microarray.

� Based on the RNA hybridisation data and the signals of all oligonucleotides representinga gene, each gene is evaluated for its expression.

� For expressed genes the best 2-3 oligonucleotides are selected for the final MyArraydesign.

� The selection of the best oligonucleotides for genes that are not detected in the RNAsample is made on the basis of the DNA hybridisation data.

� The MyArray design contains 2-3 oligonucleotides for each gene that are optimal fora high-quality gene expression analysis.

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Figure 1 – MyArray strategy. An initial large microarray design is developed. A complex RNAsample and a genomic DNA sample are hybridised to those microarrays. Based on RNAhybridisation data the best performing oligonucleotides are selected for expressed genes. Fornon-expressed genes, DNA hybridisation data are used to select the best suited oligonucleotides.The final MyArray design allows for high quality and cost-efficient gene expression analysis.

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2 Recommendations for Samples

Before submitting samples to OakLabs GmbH for MyArray service, please carefully readthrough the guidelines below.

RNA Samples

The RNA template is a very important component of our MyArray service. We compiledrecommendations to assure high quality results.

� The selection of the best oligonucleotides is preferably done based on the RNA hy-bridisation data due to the significantly higher signals compared to DNA hybridisationdata. Therefore the RNA sample should contain as many transcript species as possible.Using a pool of RNA samples from different tissues, treatments or stages, is highlyrecommended.

� We generally recommend a column-based method for isolation of total RNA (e.g.Qiagen or Macherey&Nagel).

� If you use phenol for isolation, we highly recommmend a column-based purification ofthese samples prior to microarray analysis.

� We recommend a DNase digestion if considerable amounts of genomic DNA are present.DNA can be either digested on column or in solution. The latter requires an additionalcolumn based purification.

� RNA should be eluted in RNase-free water or in a low-salt buffer; drying down RNAcompletely should be avoided.

� Avoid DEPC treated water for resuspension of RNA since DEPC can inhibit enzymaticreactions.

� Check the RNA sample by running an agarose gel containing formaldehyde or by usingthe Bioanalyzer (Agilent Technologies). The 28S:18S rRNA ratio for eukaryotes andthe 23S:16S rRNA ratio for prokaryotes should be >1. The software of the Bioanalyzerallows the generation of an RNA Integrity Number (RIN) to check integrity and overallquality of total RNA samples. The RIN value is calculated by a proprietary algorithmwhich analyses the entire electrophoretic trace of a sample enabeling an universal andunbiased comparison of samples. A RIN number of 10 indicates high RNA quality, anda RIN number of 1 indicates low RNA quality. RNA quality for gene expression profilingexperiments should be as high as possible and samples that are to be compared shouldhave similar RINs (delta-RIN <1). According to published data and our own experiencewe recommend a RIN ≥ 6 to proceed with gene expression analysis (see also Fleige,S. et al. (2006) Mol Aspects Med. 27:126-139).

� Measure the OD 260/280 and OD 260/230 ratios which indicate contaminations withprotein, salt and solvents; both ratios should be greater than or equal to 1.8.

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� Store RNA at −20 ◦C for short-term or −80 ◦C for long-term.

� At least 500 ng total RNA is required for RNA quality control and microarrayanalysis.

� Ideally, the total RNA sample has a concentration of 20 - 50 ng/µl.

DNA Samples

Even if your RNA sample is very complex it is unlikely that transcripts of all genes will bedetected. In order to select well performing oligonucleotides also for transcripts that are notin the RNA sample you provide, we hybridise genomic DNA to a second microarray. Basedon the DNA hybridisation data we select the best oligonucleotides for the genes where aselection based on the RNA hybridisation was not possible.

� DNA should be of high molecular weight.

� We recommend a RNase digestion if considerable amounts of RNA are present. Afterdigestion a column based purification is required.

� Samples may be suspended in a wide variety of buffers, including 10 mM Tris, water,or Qiagen EB. Please make sure that buffers contain no more than 0.1 mM EDTA.

� At least 3 µg genomic DNA is required, ideally the sample has a concentration ofabout 100 ng/µl.

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3 Sample Shipping

� Complete the sample submission form (.xls) and send an electronic copy to [email protected].

� Make sure your reaction tubes are properly labelled and sample IDs on the tubes matchsample IDs in the sample submission form; consider that labels and ink might comeoff at low temperature.

� Put your samples in a suitable box to make sure the tubes do not break and caps donot open during shipping.

� Ship your samples on a sufficent amount of dry ice (prefered), consider weekends andholidays that might cause delays in shipping. Alternatively, your samples can also beshipped preserved in RNAstable (Biomatrica) at ambient temperatures.

� Ship to the following address:

OakLabs GmbH

Neuendorfstr. 16b

16761 Hennigsdorf

GERMANY

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