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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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. Further elucidating these mechanisms will be useful for increasing seed yields in crops. Seeds develop in plant ovaries after fertilization, and they mature through a series of developmental stages^1,6,7. Further understanding the molecular mechanism underlying seed development requires detailed, precise gene expression profiles from a series of developing seeds to be produced. However, the high amounts of oils, proteins, carbohydrates, and polyphenols in plant seeds make it difficult to isolate highly purified RNA, which precludes precise profiling of gene expression.

Here, we introduce an efficient method for RNA isolation from oilseeds containing large amounts of oils, proteins, and polyphenols. Using this method, researchers will be able to prepare highly purified RNA. Such RNA will be useful for monitoring transcriptional changes in key genes controlling the metabolic regulation of seed storage reserves in developing and mature oilseeds.

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Protokół

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. Place fruits on aluminum plates kept at 4 °C and isolate seeds from the fruits under a stereo microscope.
   1. Place the isolated seeds (approximately 200 seeds) into 1.5 mL polypropylene tubes that have been stored in an aluminum rack on ice and immediately place the tubes in liquid nitrogen.
4. Remove the tubes from the liquid nitrogen and return them to the aluminum rack on ice. Add 100 µL of the buffer containing 1% PVP and centrifuge for 1 min at 1,000 x g at 4 °C.
5. Homogenize the sample with a stainless steel pestle using a motor-grinder for 60 s while keeping the tube in the aluminum rack on ice.
6. Add 550 µL of the buffer containing 1% PVP, mix the buffer gently by turning the tube upside down, and incubate for 10 min at 25 °C.
7. Centrifuge for 5 min at 8,000 x g at 25 °C and transfer 550 µL of the supernatant to a new 1.5 mL polypropylene tube.
8. Centrifuge for 5 min at 10,000 x g at 25 °C and transfer 450 µL of the supernatant to a new 1.5 mL polypropylene tube.
9. Use the supernatant as the cell lysate for RNA extraction. Hereafter, follow the procedure described in the manufacturer's instructions in the commercially available kit.
10. Elute the RNA using a minimum volume of the elution buffer to ensure a sufficiently high concentration of RNA for monitoring the expression patterns of low level transcripts. Store the RNA in a deep freezer until used.

2. Verification of RNA Quality

1. Thaw the total RNA, mix by gentle tapping, and place the tube in an aluminum rack on ice.
2. Measure the RNA concentration, A260/A280 ratio, and A260/A230 ratio using a microvolume spectrophotometer.
3. Dilute the total RNA in RNase-free water to a final concentration of 70 ng/µL.

3. Reverse Transcription of Total RNA from Seeds

1. Thaw the total RNA and buffer sets from the commercial kit. Keep the enzymes from the kit on ice after gentle tapping. Prepare nuclease-free 0.2 mL polypropylene tubes.
2. Add 5 µL of total RNA, 1 µL of Oligo dT primer (50 µM), and 1 µM of Random 6-mer (50 µM) to the 0.2 mL polypropylene tube on ice.
3. Incubate for 15 min at 37 ºC and place on ice.
4. Hereafter, follow the procedure described in the manufacturer's instructions in the reverse transcription-PCR kit.
5. Place the reverse transcription products on ice before use.

4. Quantitative Real-time PCR Analysis

1. Construct plasmids harboring the target gene sequences using the manufacturer's protocol.
2. Adjust the concentrations to 100-500,000 copies/µL for DNA templates for the standard curves.
3. Dilute the cDNA solutions (1:100) with distilled water.
4. Add 2 µL of the diluted cDNA solutions and the plasmids for the standard curves to the master mix from the quantitative real-time PCR kit.
5. Set up real-time PCR using the following cycling conditions: 95 °C for 30 s and then 40 cycles at 95 °C for 5 s and 60 °C for 35 s.
6. Analyze the copy numbers according to the manufacturer's instructions for the real-time PCR system.

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Wyniki

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).

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Dyskusje

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...

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Materiały

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