Abstract: Many experimental techniques related to molecular biology such as the Northern blot analysis, RNA protection assays, in situ hybridization and RT-PCR need maximum-yielded, highly purified RNA samples. Researchers who study with RNA extraction are well known for being extremely sensitive about their chemical solutions, equipment, glassware and even laboratories. Taking certain precautions, which are given in protocol, is more convenient than obtaining a low 260/280 spectrophotometric ratio or a blank line in RT-PCR gel for ecologic studies. In the improved protocol, which is presented here, each step was tested many times and high-quality total RNA was determined. Using this protocol, 260/280 spectrophotometric ratio was found between 1.88-1.97 and total RNA was found between 174.6-184.3 µg RNA g cell FW-1 in tobacco cells. However, in order to obtain RNA with high purity levels, an additional step may be necessary to eliminate contaminating DNA depending on the sample characteristics.