Does pooling RNA count as replication?
NO! A common practice involves pooling RNA from several experimental units (e.g. animals) in an effort to achieve more representative results. While pooling RNA in a replicated experiment may indeed improve statistical power and precision due to less variation across samples, pooling RNA is not a substitute for replicating an experiment. For instance: The practice of pooling RNA does not in itself provide a way to characterize random variation in the experiment, so replication is still needed to distinguish real differences. Pooling RNA can distort results if, for example, a particular experimental unit is problematic and contributes a misleading expression pattern that skews the results. Pooling RNA precludes the investigator from observing potentially interesting patterns in behavior across different animals or other experimental units.
