Microarray Core RNA Guide
Direct communication with a member of the core is required before sending samples. Please contact us at SScArrayCore@gmail.com before sending samples
Skin tissue samples: Skin collection protocol should be followed and samples stored at -80°C until shipped. Samples should be shipped on dry ice. Never allow samples to thaw before you send them to us.
RNA samples: All RNA samples submitted directly to the Microarray Core, by the user, must first go through sample quality control to determine RNA integrity, RNA concentration, and overall purity of the sample. Any sample that does not pass QC will be returned to the submitter.
Cells: Cells should be freshly harvested, RNA prepared immediately and samples stored at -20 degrees or colder. Never let samples thaw before you send them to us (on dry ice).
Preferred RNA preparation methods
• Qiagen RNeasy for fibrous tissue for skin
• Qiagen RNeasy for cells.
• Trizol, Phenol-Chloroform-Ethanol Precipitation
• PBMCs should use the methods in Pendergrass et al 2010 PLoS ONE.
Skin tissue samples: Please provide us with at least 4mm sized biopsies stored as described in the Sample Collection Protocol in order for us to extract sufficient RNA for analysis.
Total volume and concentration of RNA: 15 – 200 ng in a total volume of 10 ul. A higher concentration of RNA gives better and more consistent results.
Cells: In general we discourage you from sending us frozen cell pellets as they are best processed immediately after harvest. Please provide us at least half million cells in order to extract sufficient RNA for testing. Please email or call to discuss this option.
If you have any quality control data (Nanodrop, bioanalyzer, gel electrophoresis), please send a copy with your sample(s). RNA quality is the single most important factor in determining the outcome of microarray experiments. The total RNA that you submit should be intact and free of protein and DNA contamination.
To ensure samples were not damaged in transport, we will perform QC here on a Thermo Scientific NanoDrop 2000 and analyze integrity on an Agilent Bioanalyzer. We will look at concentration as well as 260/280 and 260/230 ratios, and RNA integrity before proceeding.
Samples that do not meet quality control standards will be flagged and QC data distributed to the submitter. RNA that does not pass QC will incur basic costs associated with the QC process.
Samples should meet the following minimum basic criteria:
260/280 > 2.0
260/230 > 1.8
Mass of RNA > 15 ng in a total volume of 10 microliters.
RNA integrity: RIN > 7
Explanation of parameters:
260/280 ratio: The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of RNA. A ratio of 2.0 or greater is accepted as “pure” for RNA. If the ratio is lower, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.
260/230 ratio: This ratio is used as a secondary measure of nucleic acid purity. The 260/230 values for “pure” nucleic acid are 1.8 or higher. If the ratio is lower than expected, it may indicate the presence of contaminants. Nucleic acids only absorb light that has a wavelength of 260 nm. Contaminants like phenol, buffer, ethanol, proteins and solvents, used in RNA extraction, absorb light of a 230 nm. Samples with a low 260/230 (below about 1.8) have a significant presence of these organic contaminants that may interfere with other downstream processes including microarray experiments, lowering their efficiency. We strongly recommend limiting microarray experiments to those samples with a 260/230 ratio greater than 1.8. Running samples with 260/230 ratios lower than 1.8 could have reduced yield and substantially less optimal results. Users who wish to run samples that have organic contamination indicated by a low 260/230 ratio must assume responsibility for the performance of their samples. In order to run samples with low 260/230 ratios, we will request that you sign off on the samples showing you are aware of the risk associated with performing microarray experiments with samples that do not the meet sample purity standards validated by the Microarray Core.
RNA integrity: Integrity of RNA starting materials is a critical step in gene expression analysis. Using the Agilent 2100 bioanalyzer with RNA 6000 Nano Kit, quality is determined. Profiles generated on the analyzer yield information on concentration, allow a visual inspection of RNA integrity, and generate ribosomal ratios. The integrated RNA integrity number (RIN) algorithm removes user-dependent interpretation of quality. The higher the RIN the more intact the RNA. RIN ranges from 1 (totally degraded RNA) to 10 (completely intact RNA). Our validation tests show that an RIN number greater than 7 correlates with successful downstream microarray experiments. It is important to note that the RIN does not indicate the usefulness of gene expression data only the successful measurement of the data.
Sending samples: an order request must be submitted to us and a shipping log, detailing sample information, must accompany the shipment. Failure to provide these could delay processing.
Address: Michael Whitfield
Attn: Tammara Wood
Dartmouth Medical School
Department of Genetics
Vail Loading Dock
Hanover, NH 03755
Turn-around Time: Given the time required for the completion of RNA extraction and/or analysis, microarray labeling and hybridization and initial data analysis, the turn-around time will be at least 2 weeks from the time you submit your samples. If we have a backlog which may occur due to large numbers of samples being submitted in short time period, we will inform of the delay and our estimated time to complete the analysis.