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). However there are several other methods/kits available, and you should try and find one that is good for your particular material. If total RNA is to be used for miRNA analysis, it is important to make sure the purification method retains low molecular weight species.
The amount of total RNA required for gene array analysis depends on which assay is to be used. 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 (for Whole Transcript arrays, such as Clariom and Gene ST arrays.) 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 (for Whole Transcript arrays, such as Clariom and Gene ST arrays) enables whole transcript analysis of small amounts of total RNA. View datasheet. Input range for fresh-frozen cells is 100 pg-2 ng total RNA (validated for HeLa cells). The kit can also be used for 500 pg-50 ng total RNA extracted from FFPE samples (1 to 9 year old samples have been validated). 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 customers to use the Affymetrix RNA Quantification Kit. We recommend analysis of 12 or 30 samples as arrays and reagents are sold in packs of 12/30. For other numbers, please contact us. Always contact us before sending any samples.
- The 3' IVT-PLUS protocol (for GeneChip 3' Expression Arrays) 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.
- For Prokaryotic samples, please contact us.
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 expression studies needs to be pure, intact and of even quality. 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. To avoid amplification of contaminating DNA (due to random priming), DNase-treatment is recommended. There are RNA-kits available that include DNase-treatment in the protocol. Integrity should be measured on 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.
Presently (May 2017) we keep the GeneChip WT PLUS Reagent Kit (for Whole Transcript arrays, such as Clariom and Gene ST arrays) in stock. For all other assays, please inquire about which minimum order size we can accept, as it will vary depending on pack sizes of reagent kits and arrays.
Please observe that the reagent kit for preparing samples for Gene ST and Exon Arrays was changed in 2014. Any customer who wants to combine new data with data generated before the change, is kindly asked to contact us for information regarding comparability.
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!
For large scale studies (more than hundred samples), please contact us!
Transport to SCIBLU Genomics
Samples should be transported on dry ice. Please contact us before sending your samples to make sure that someone will receive the package. You will be placed in queue upon sample arrival.
SCIBLU Genomics, Microarray unit
Inst. för Immunteknologi (Byggnad 406)
Medicon Village, Byggnad 402A Scheelevägen8