NASC's Affymetrix Service

Some sample preparation hints

Here are some things to bear in mind when preparing samples for sending to an Affy service. Most of these are equally relevant to animals and plant species.

  • If we prepare RNA in-house, we extract and prepare our RNA samples using RNAeasy extraction kits from Qiagen. This has worked for all kinds of material from Arabidopsis, through Aloe to Rubber tree and is usually a good starting point. We have also used commercial kits for blood RNA extraction from horse. If in doubt use a commercial kit, the stuff you will do downstream is too expensive to mess around with home-brew protocols at this stage. For difficult tissues such as seed see below.
  • We grow our samples under continuous (24 hour) light conditions to reduce circadian influences on the materials. All stocks grown at NASC since 1999 have been bulked under 24-hr light conditions. This also helps to reduce potential maternal circadian effects in seeds grown from NASC stocks. This is harder in animals but please try to reduce environmental variability where possible.
  • In addition to growth under 24hr light conditions, materials are collected at defined, consistent, and recorded times of day to reduce any material or environmental circadian effects.
  • Material is also collected under defined and recorded light and temperature conditions. Handling should be minimal and each individual plant, seedling or animal explant should be snap-frozen immediately upon harvesting to reduce any stress induction of transcripts upon handling. For samples collected in the field we have successfully used RNAlater from Ambion (e.g. Rubber tree samples from Kew Palm house in Spring 2010).
  • Plants and animals should be selected at a clearly identified numeric stage as defined in Arabidopsis by Boyes et al, (2001). Also see our plant ontologies help page. Individuals should be individually hand picked to align with developmental stagerather than mass harvesting by chronological time. As shown in the reference above, chronological time is a poor determinant of developmental stage for analysis of mutants and comparisons between individuals.
  • Light, humidity and a stereotyped watering/feeding regime throughout the growing period should be followed and recorded.
  • Pooling is an individual choice, it reduces sensitivity but can equalise biological variability. If you decide to pool then make sure that all the samples are truly equivalent.
RNA Extraction Protocols

The standard RNA extraction protocols for microarray analysis that most of our customers use are Qiagen and Trizol protocols. Our experience at NASC shows that these protocols are not always ideal for a small number of plant tissues. Below are a list of additional extraction protocols for certain plant tissues;