Short Communication

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

Published: January 24, 2013
Genet. Mol. Res. 12 (1) : 223-229 DOI: 10.4238/2013.January.24.14

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 A260/A280 and A260/A230 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 β-actin as an internal control. We concluded that the total RNA isolated by this protocol is of sufficient quality for molecular applications.

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 A260/A280 and A260/A230 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 β-actin as an internal control. We concluded that the total RNA isolated by this protocol is of sufficient quality for molecular applications.