Comparison of Different Methods for RNA Extraction from Floral Buds of Tree Peony (Paeonia suffruticosa Andr.)

Yan Gao,Yao Song,Guangqi Zhao,Kang Ye,Changhua Jiang,Shucheng Feng

doi:10.15835/nbha44210402

Yan Gao, Yao Song + Show 4 more

Open Access

https://doi.org/10.15835/nbha44210402

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Abstract

Tree peony (Paeonia suffruticosa Andr.), a species native to China, is one of the most important ornamental and medicinal plants. Like other tree species in temperate and boreal zones, the dormancy-activity transition of floral buds is critical for blooming time and fruit production. However, floral buds contain high levels of secondary metabolites, making the isolation of high quality RNA difficult. To obtain a method suitable for extracting RNA from floral buds of tree peony, we evaluated five different methods, including the cetyltrimethylammonium bromide (CTAB) and Sodium dodecyl sulfate (SDS)-based methods, a modified SDS-TRNzol protocol, and two commercial kits (TRNzol and Qiagen RNeasy Plant Mini Kit). The modified SDS-TRNzol method was capable of efficiently removing polyphenols and other metabolites in floral buds. The isolated RNA was of high purity and integrity, as demonstrated by the the A260/280 ratio of approximately 2.0, and RIN values of more than 9.0. Gel electrophoresis analysis indicated that the extracted RNA had clear 28S and 18S ribosomal RNA bands without DNA contamination. The RNA isolated by this protocol was successfully used for downstream manipulations, such as RT-PCR, RACE, and real-time PCR. Together, the modified SDS-TRNzol protocol is an easy, efficient, and highly reproducible method for RNA isolation from floral buds rich in secondary metabolites.

Highlights

The floral industry has comprised an important part of the global economy, featured as cultural and high-added values
To obtain a method suitable for extracting high quality RNA from secondary metabolites-rich floral buds of tree peony, we evaluated five different methods, including the cetyltrimethylammonium bromide (CTAB) (Chang et al, 1993) and Sodium dodecyl sulfate (SDS)-based methods (Zhou et al, 1999; Wu et al, 2006), a modified SDS-TRNzol protocol (Wang et al, 2012), and two commercial kits (TRNzol and Qiagen RNeasy Plant Mini Kit)
Floral buds are rich in polysaccharides, polyphenols, and other secondary metabolites, which interfere with RNA extraction and downstream applications

Summary

Introduction

The floral industry has comprised an important part of the global economy, featured as cultural and high-added values. Floral buds often contain high levels of carbohydrates, anthocyanidin, polyphenols, hormones, and other secondary metabolites (Mornya et al, 2011; Li et al, 2012). To obtain a method suitable for extracting high quality RNA from secondary metabolites-rich floral buds of tree peony, we evaluated five different methods, including the cetyltrimethylammonium bromide (CTAB) (Chang et al, 1993) and Sodium dodecyl sulfate (SDS)-based methods (Zhou et al, 1999; Wu et al, 2006), a modified SDS-TRNzol protocol (Wang et al, 2012), and two commercial kits (TRNzol and Qiagen RNeasy Plant Mini Kit). We present an effective and rapid protocol based on the combination of SDS and TRNzol, allowing isolating high quality total RNA from floral buds of tree peony

Materials and Methods

Results and Discussion

Method

Conclusions