The following information should act as a guide for important steps in designing a microarray experiment. It is not intended to cover every aspect of an experiment but rather to give suggestions for the major steps involved.
Important aspects to monitor are the collection, handling and storage of the samples. This is particularly true in studies involving human patients or animal studies where samples are collected in a clinic or operating room.
Sample Collection of Nucleic Acids
It is critical that tissue samples be stabilized in terms of RNase activity immediately following their isolation from the host. RNase is a small, very active enzyme that cannot be inactivated by autoclaving. All methods of RNase stabilization inactivate enzymatic activity but the enzyme will come back to life (refold into an active state) easily when conditions become favorable again. Although DNA degradation can be a problem as well, DNase requires Ca+2 as a co-factor for activity and is not nearly as robust as RNase that requires no co-factor for activity.
Most importantly, do NOT let the tissue sample linger at room temperature following isolation from the organism of origin. The nucleic acids will begin to breakdown immediately following removal from the organism, especially the RNA. The breakdown process is rapid and thorough. A good way to avoid this, when it is not possible to process samples immediately upon collection is to use a product called RNAlater (Qiagen or Ambion). This solution will stabilize the RNA within the tissue at room temperature for a few hours or at 4°C or -20°C for longer periods of time. Large tissue samples need to be cut into thin slices, consult the manual for directions on proper handling.
Macromolecule Isolation of Nucleic Acids
There are 3 choices for inactivating RNase during RNA isolation:
If your tissue is small or can be cut into thin slices, RNAlater is a good choice for stabilizing the RNA in the tissue as mentioned above.
Tissue can be added to a guanidinium saturated phenol solution such as Trisol, TriReagent, RNAsol, etc. and homogenized immediately following extraction from the subject using either a Polytron, mini-homogenizer or bead beater. The resulting homogenate can be stored at -80°C for long periods of time prior to the isolation of RNA or DNA.
The tissue can be flash frozen in liquid nitrogen and stored at -80°C. For some tissues, where a LN2 frozen powder is required prior to nucleic acid isolation, this may be preferable. A phenol-based reagent is recommended for the final nucleic acid isolation here as well.
Regardless of which of the above three methods are employed, it is important to couple the homogenization process to a final column-based RNA isolation step. Using the homogenization reagent alone will carry over a lot of DNA and may not remove all the proteins, RNA or DNA binding proteins in particular, on the first pass. Using a column will get rid of these carryover contaminants.
Handling and Storage of Nucleic Acids:
Although not as sensitive to freeze/thaw cycles as proteins, it is not a good idea to freeze and thaw nucleic acid samples too many times. If possible, aliquot them early on and at a concentration that is high enough to perform any assay requiring high concentrations. Further, nucleic acids store better at higher concentrations (e.g., 0.25 – 1.0 µg/µl).