An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Summary

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

Abstract

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

Introduction

Plants produce seeds, which give rise to the next generation. Seeds accumulate large amounts of storage reserves, such as oils, carbohydrates, and proteins, for post-germinative growth. Humans utilize seed storage reserves as sources of food and animal feed, and thus plant seeds are one of the major suppliers of edible organic matter worldwide. Increasing seed yields is an important challenge in plant science.

Since seed storage reserves are commercially valuable sources of food and industrial materials, the molecular mechanisms underlying the regulation of the metabolism of these reserves have been widely investigated^1-6. Furthe....

Protocol

1. Extraction of Total RNA from Plant Seeds

1. Prepare buffer sets, spin columns, 1.5 and 2.0 mL polypropylene tubes, and nuclease-free 1.5 mL polypropylene tubes.
2. Add 1% (w/v) molecular biology grade polyvinylpyrrolidone (hereafter referred to as PVP) to cell lysis buffer for RNA extraction and vortex vigorously. Incubate for 20 min at 25 °C to dissolve completely. After 20 min incubation, mix the buffer gently by turning the tube upside down to prevent the formation of bubbles. Store at room temperature (15-25 °C) before use.
3. Harvest fruits from Arabidopsis thaliana plants and place in 1.5 mL polypropylene tubes on ice....

Results

We first investigated the optimal concentration of PVP using Arabidopsis mature seeds. Total RNA was isolated from approximately 1,000 seeds according to the protocol described above using cell lysis buffer containing 0%, 0.25%, 0.5%, 1.0% or 2.0% PVP. After homogenization and centrifugation, the supernatant was collected while avoiding the oil layer and seed debris (Figure 1A).

Discussion

Gene expression profiles contribute to our understanding of plant physiology; therefore, specific RNA isolation methods have been developed for each sample condition^9-12. We investigated the processes that were inhibited during RNA isolation from seeds and found that RNA binding to silica membranes was severely inhibited. Large amounts of oil, proteins, and polyphenols inhibit RNA isolation. We modified the RNA extraction process to remove these compounds with a lysis solution before the process of RNA binding.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RNeasy Plant Mini Kit	QIAGEN	74904
polyvinylpyrrolidone	Sigma-Aldrich	P5288-100G
HOMOGENIZER S-303	AS ONE	1-1133-02
NanoDrop Lite	Thermo Scientific	ND-NDL-US-CAN
PrimeScript RT reagent Kit (Perfect Real Time)	TAKARA	RR037A
KAPA SYBR Fast qPCR kit	Kapa biosystems	KK4601