Abstract: Total RNA was extracted from immature seed of Arabidopsis thaliana and Nicotiana tobaccum using four different methods . Trizol reagent resulted in low quantity and poor quality of RNA. The other three methods could isolate RNA from immature seed with quality that match the downstream experiments. Among these methods, CTAB-Isopropyl alcohol method could get the highest yield of total RNA, with the advantages of time saving and low cost. Bioteke kit is also a convenient method to isolate immature seed RNA under room temperature. CTAB-LiCl method could get higher quality total RNA without genome DNA contamination. However, LiCl couldn’t precipitate small molecule RNA well. The quality of total RNA isolated using the above three methods was analyzed with UV spectrophotometer and agarose gel electrophoresis. The OD260/OD280 ratio was 1.80-1.97 and OD260/OD230 ratio was 2.03-2.37. 28S rRNA and 18S rRNA were clearly detected in the RNA electrophoresis. Total RNA isolated by these methods were reverse transcribed and high quality cDNAs were obtained.