Efficient isolation of high-quality RNA from lotus Nelumbo nucifera ssp nucifera tissues.

ABSTRACT

Nelumbo nucifera is widely used as food, as an ornamental, in medicine, and as packing material; it is also reported to have anti-HIV effects and antioxidant capacity. We sought an improved method for extracting high-quality total RNA from different tissues of N. nucifera. Four methods for RNA extraction were assessed for their ability to recover high-quality RNA applicable for evaluation of polyphenol oxidase (PPO) gene expression profiles. The recovery and quality of the RNA obtained from five different tissues by the best CTAB-LiCl method were evaluated through UV light absorbance. Both A(260)/A(280) and A(260)/A(230) absorbance ratios were more than 2.0; the yield ranged from 59.87 to 163.75 µg/g fresh weight. The brightness of the 28S band was approximately twice that of 18S; the latter was also considered as high-quality RNA. The PPO gene fragment (606 bp) was successfully amplified by RT-PCR, demonstrating the integrity of the isolated RNA. The relative expression levels of the PPO gene based on RT-PCR in five tissues of lotus were rhizome buds (2.66), young leaves (2.42), fresh cut rhizome (2.02), petals (1.80), and petiole (1.65), using housekeeping gene ß-actin as an internal control. We concluded that the total RNA isolated by this protocol is of sufficient quality for molecular applications.