Highly sensitive techniques for transcriptome analysis, such as microarrays, complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs), and others currently used in functional genomics require a high RNA quality and integrity, as well as reproducibility among extractions of replicates from the same tissue. There are, however, few technical papers comparing different homogenization techniques and reagents to extract RNA from small quantities of plant tissue. We extracted RNA from tomato seedlings with the three different commercial reagents TRIZOL LS®, TRIZOL®, and TRI Reagent® in combination with pulverization, homogenization-maceration in a mortar, and homogenization with mild vibration plus glass beads, and evaluated total RNA integrity-quality and yield. Pulverization under liquid nitrogen combined with TRIZOL LS® as extraction reagent and homogenization-maceration in mortar with TRI Reagent®, are the procedures that rendered higher RNA yield, integrity and quality, as well as reproducibility among independent RNA extractions. In contrast, short mild vibration pulses (4500 r.p.m. for 5 s) mixed with glass beads, rendered low extraction efficiency and caused, in most cases, partial RNA degradation.