Design of Yeast Genomic Expression Experiments

III. Sample collection

A. Be as controlled as possible! Collect all samples as identically as possible

B. Collect by centrifugation (3-5 min at ~3g) or filtration (<1 min by filtering culture

over a sterile 0.45 um filter and collecting entire filter)

C. Again, few variables

1. Collect cells ~experimental temperature to avoid

temperature shock

2. Collect cells as quickly as possible

3. Do not wash or handle cells unnecessarily

D. Example problems:

1. washing cells induces many variables and can induce the stress response

( induces changes in nutrients, osmolarity, ionic, pH, etc.)

2. collecting cells on ice can induce cold shock

3. lengthy collection can induce hypoxia

4. variable collection time is Very Bad!

V. Microarray hybridization

A. Again, as controlled as possible

1. For optimal comparison within a timecourse, perform sample collection, RNA

preparation and labeling, and especially hybridizations together for consistency

B. Always be consistent with labeling and hybridizations (time, temperature) to improve

reproducibility

VI. Duplicate experiments