Pools
Pooling samples can be an excellent way to probe for answers in situations where your RNA amounts are low. However, great care should be taken when setting up the pools. When choosing which samples to pool, follow the same guidelines as you would for choosing replicates. Keep your pool size consistent - comparing a two-sample pool with a four-sample pool is not as useful as comparing two three-sample pools. Finally, you still need replicates! Four pools of three animals only yields four data points, not twelve. Even if your samples are pooled, you still need replicates to be able to apply statistical tests.