The amount of total RNA required for a microarray analysis depends on which method will be used to prepare the samples. For data comparability the input amount should be equal for all samples within a project. Below is a guide to the most commonly used assays. For other assays, please inquire.
- The WT Plus protocol requires 100-500 ng high quality total RNA. Please provide a minimum of 500 ng in RNase-free water, concentration 75-100 ng/µl. If < 300 ng is available, total RNA should be delivered in 5-10 µl RNase-free water.
- The WT Pico protocol enables whole transcript analysis of small amounts of total RNA. Input range for fresh-frozen cells is 100 pg-2 ng total RNA. The kit can also be used for 500 pg-50 ng total RNA extracted from FFPE samples. Maximum sample volume is 3 µl. Samples should be DNase treated and carriers must not be used when extracting total RNA. Please read the manual p 1-13 carefully. To assess low concentrations we recommend using the Affymetrix RNA Quantification Kit.
- The 3' IVT-PLUS protocol requires 100-500 ng high quality total RNA. Please provide a minimum of 500 ng in RNase-free water, concentration 100-200 ng/µl. If < 300 ng is available, total RNA should be delivered in 5-10 µl RNase-free water.
- RNA concentration should always be checked after dilution to the appropriate concentration. Samples should be delivered in eppendorf tubes or PCR strips and labelled individually with a marker pen directly on the lid (no tape or paper labels).
Total RNA for microarray expression analysis needs to be pure, intact and of even quality.
- Total RNA from several eukaryotic species can be isolated using TRIzol® Reagent, (Invitrogen) followed by purification with RNeasy® Mini Kit (Qiagen). Another useful kit is ZYMO, Direct-zol RNA Mini Prep (Nordic Biolabs). There are several other methods/kits available, please use one that is appropriate for your particular material.
- To avoid amplification of contaminating DNA, DNase-treatment is recommended. There are RNA-kits available that include DNase-treatment in the protocol
- If total RNA is to be used for miRNA analysis, it is important to make sure the purification method retains low molecular weight species.
- We recommend using the NanoDrop for determining the sample concentration and purity. OD260/280 ratio should be above 1.8 and OD260/230 preferably close to 2.0.
- RNA integrity should be measured using an Agilent Bioanalyzer and RIN values preferably be above 7.
- All NanoDrop and Bioanalyzer testing of total RNA at SCIBLU Genomics will be charged according to our Quality Control pricelist. Customers wishing to buy the quality control service must provide 200 ng extra total RNA.
It is vital to design experiments in such a way that non-informative and random variations are eliminated. Samples that are to be compared to each other should be run simultaneously in the laboratory. Depending on the source of RNA the recommended number of biological replicates will vary. Most experimental designs need three to six replicates per group in order to produce meaningful data. Ask us and we will advice you!
Samples should be transported on dry ice. Please contact us before sending your samples to make sure that someone will receive the package. Send the samples to the following address:
SCIBLU Genomics Microarray unit
Inst. för Immunteknologi (406)
Medicon Village, byggnad 402A
SE 223 63 Lund