Guidelines for Sample Submission to OakLabs GmbH Gene expression profiling on Agilent microarrays Version 4, May 2014 For further information, please contact: Email: [email protected] Phone: +49 33 02 2 89 79 69 Contents 1 Recommendations for RNA Samples 2 2 RNA Sample Requirements 2 3 RNA Sample Shipping 3 4 Your RNA Samples at OakLabs 3 1 Agilent expression profiling, May 2014 www.oak-labs.com Before submitting RNA samples to OakLabs GmbH for labelling and hybridisation, please carefully read through the guidelines below. 1 Recommendations for RNA Samples The most important component of an expression profiling experiment is the RNA template. We have compiled recommendations to assure high quality results. 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 of these 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 additional column based purification. RNA should be eluted in RNase-free water or in a low-salt buffer; drying down RNA completely should be avoided. Avoid DEPC treated water for resuspension of RNA since DEPC can inhibit enzymatic reactions. Check each RNA sample by running an agarose gel containing formaldehyd or by using the Bioanalyzer (Agilent Technologies). The 28S RNA/18S RNA ratio for eukaryotes and the 23S RNA/16S RNA ratio for prokaryotes should be >1. The software of the Bioanalyzer allows the generation of an RNA Integrity Number (RIN) to check integrity and overall quality of total RNA samples. The RIN value is calculated by a proprietary algorithm which analyses the entire electrophoretic trace of a sample enabeling an universal and unbiased comparison of samples. A RIN number of 10 indicates high RNA quality, and a RIN number of 1 indicates low RNA quality. RNA quality for gene expression profiling experiments should be as high as possible and samples that are to be compared should have similar RINs (delta-RIN <1). According to published data and our own experience we 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 with protein, salt and solvents; both ratios should be greater than or equal to 1.8. Store RNA at −20 ◦ C for short-term or −80 ◦ C for long-term. 2 RNA Sample Requirements At least 500ng total RNA microarray analysis. per sample is required for RNA quality control and 2 Agilent expression profiling, May 2014 www.oak-labs.com Ideally, total RNA samples have a concentration of 20 - 200 ng/µl. If required, smaller amounts of starting material can also be processed for microarray analysis. Please inquire prior to sample submission. 3 RNA 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 match sample IDs in the sample submission form; consider that labels and ink might come off at low temperature. Put your samples in a suitable box to make sure the tubes do not break and caps do not open during shipping. Ship RNA on a sufficent amount of dry ice (prefered), consider weekends and holidays that might cause delays in shipping. Alternatively, RNA can also be shipped preserved in RNAstable (Biomatrica) at ambient temperatures. Ship to the following address: OakLabs GmbH Agilent Service Neuendorfstr. 20b 16761 Hennigsdorf GERMANY 4 Your RNA Samples at OakLabs Once we have received your RNA samples we complete the RNA quality control within one week. By default RNA quality check is performed using the Bioanalyzer and quantity is measured using the Nanodrop photometer. If your samples pass our quality control we proceed with labelling. If one or more of your samples fails our quality control, we consult with you how to proceed. After succesful quality control, we usually perform labelling and hybridisation within two weeks. Data are delivered via FTP o r on a USB stick by mail, depending on your preference. 3