We thank the staff of Functional Genomics Facility and&#160; Spectrography and Bioimaging Facility, NIBB Core Research Facilities, and Model Plant Research Facility, NIBB Bioresource Center.

1. Extraction of Total RNA from Plant Seeds
<ol>
	<li>Prepare buffer sets, spin columns, 1.5 and 2.0 mL polypropylene tubes, and nuclease-free 1.5 mL polypropylene tubes.</li>
	<li>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 &#176;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 &#176;C) before use.</li>
	<li>Harvest fruits from Arabidopsis thaliana plants and place in 1.5 mL polypropylene tubes on ice. Place fruits on aluminum plates kept at 4 &#176;C and isolate seeds from the fruits under a stereo microscope.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Remove the tubes from the liquid nitrogen and return them to the aluminum rack on ice. Add 100 &#181;L of the buffer containing 1% PVP and centrifuge for 1 min at 1,000 x g at 4 &#176;C.</li>
	<li>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.</li>
	<li>Add 550 &#181;L of the buffer containing 1% PVP, mix the buffer gently by turning the tube upside down, and incubate for 10 min at 25 &#176;C.</li>
	<li>Centrifuge for 5 min at 8,000 x g at 25 &#176;C and transfer 550 &#181;L of the supernatant to a new 1.5 mL polypropylene tube.</li>
	<li>Centrifuge for 5 min at 10,000 x g at 25 &#176;C and transfer 450 &#181;L of the supernatant to a new 1.5 mL polypropylene tube.</li>
	<li>Use the supernatant as the cell lysate for RNA extraction. Hereafter, follow the procedure described in the manufacturer&#39;s instructions in the commercially available kit.</li>
	<li>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.</li>
</ol>
2. Verification of RNA Quality
<ol>
	<li>Thaw the total RNA, mix by gentle tapping, and place the tube in an aluminum rack on ice.</li>
	<li>Measure the RNA concentration, A260/A280 ratio, and A260/A230 ratio using a microvolume spectrophotometer.</li>
	<li>Dilute the total RNA in RNase-free water to a final concentration of 70 ng/&#181;L.</li>
</ol>
3. Reverse Transcription of Total RNA from Seeds
<ol>
	<li>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.</li>
	<li>Add 5 &#181;L of total RNA, 1 &#181;L of Oligo dT primer (50 &#181;M), and 1 &#181;M of Random 6-mer (50 &#181;M) to the 0.2 mL polypropylene tube on ice.</li>
	<li>Incubate for 15 min at 37 &#186;C and place on ice.</li>
	<li>Hereafter, follow the procedure described in the manufacturer&#39;s instructions in the reverse transcription-PCR kit.</li>
	<li>Place the reverse transcription products on ice before use.</li>
</ol>
4. Quantitative Real-time PCR Analysis
<ol>
	<li>Construct plasmids harboring the target gene sequences using the manufacturer&#39;s protocol.</li>
	<li>Adjust the concentrations to 100-500,000 copies/&#181;L for DNA templates for the standard curves.</li>
	<li>Dilute the cDNA solutions (1:100) with distilled water.</li>
	<li>Add 2 &#181;L of the diluted cDNA solutions and the plasmids for the standard curves to the master mix from the quantitative real-time PCR kit.</li>
	<li>Set up real-time PCR using the following cycling conditions: 95 &#176;C for 30 s and then 40 cycles at 95 &#176;C for 5 s and 60 &#176;C for 35 s.</li>
	<li>Analyze the copy numbers according to the manufacturer&#39;s instructions for the real-time PCR system.</li>
</ol>

<ol>
	<li>Hills, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+storage-product+synthesis+in+seeds.">Control of storage-product synthesis in seeds.</a> Curr Opin Plant Biol. 7 (3), 302-308 (2004).</li><li>Li-Beisson, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyl-lipid+metabolism.">Acyl-lipid metabolism.</a> Arabidopsis Book. 11, e0161 (2013).</li><li>Bates, P. D., Stymne, S., Ohlrogge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+pathways+in+seed+oil+synthesis.">Biochemical pathways in seed oil synthesis.</a> Curr Opin Plant Biol. 16 (3), 358-364 (2013).</li><li>Santos-Mendoza, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+gene+regulatory+networks+that+control+seed+development+and+maturation+in+Arabidopsis.">Deciphering gene regulatory networks that control seed development and maturation in Arabidopsis.</a> Plant J. 54 (4), 608-620 (2008).</li><li>Durrett, T. P., Benning, C., Ohlrogge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+triacylglycerols+as+feedstocks+for+the+production+of+biofuels.">Plant triacylglycerols as feedstocks for the production of biofuels.</a> Plant J. 54 (4), 593-607 (2008).</li><li>Kanai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Plastidic+DEAD-box+RNA+helicase+22,+HS3,+is+essential+for+plastid+functions+both+in+seed+development+and+in+seedling+growth.">The Plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth.</a> Plant Cell Physiol. 54 (9), 1431-1440 (2013).</li><li>Kanai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+of+oil+biosynthesis+during+the+mid-phase+of+seed+development+enhances+oil+content+in+Arabidopsis+seeds.">Extension of oil biosynthesis during the mid-phase of seed development enhances oil content in Arabidopsis seeds.</a> Plant Biotechnol J. 14 (5), 1241-1250 (2016).</li><li>Dekkers, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+reference+genes+for+RT-qPCR+expression+analysis+in+Arabidopsis+and+tomato+seeds.">Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds.</a> Plant Cell Physiol. 53 (1), 28-37 (2012).</li><li>Salzman, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+RNA+isolation+method+for+plant+tissues+containing+high+levels+of+phenolic+compounds+or+carbohydrates.">An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates.</a> Plant Mol Biol Rep. 17 (1), 11-17 (1999).</li><li>Vicient, C. M., Delseny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+total+RNA+from+Arabidopsis+thaliana+seeds.">Isolation of total RNA from Arabidopsis thaliana seeds.</a> Anal Biochem. 268 (2), 412-413 (1999).</li><li>Wang, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high+quality+RNA+from+cereal+seeds+containing+high+levels+of+starch.">Isolation of high quality RNA from cereal seeds containing high levels of starch.</a> Phytochem Analysis. 23 (2), 159-163 (2012).</li><li>Birtic, S., Kranner, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high-quality+RNA+from+polyphenol-,+polysaccharide-+and+lipid-rich+seeds.">Isolation of high-quality RNA from polyphenol-, polysaccharide- and lipid-rich seeds.</a> Phytochem Analysis. 17 (3), 144-148 (2006).</li></ol>

The authors have nothing to disclose.

Gene expression profiles contribute to our understanding of plant physiology; therefore, specific RNA isolation methods have been developed for each sample condition9-12. 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 to silica membranes. The important modifications included the addition of 1% PVP (step 1.2) and the collection of the supernatant from centrifuged lysis solution (steps 1.7, 1.8). The aim of the addition of 1% PVP is to remove polyphenols. We examined the most effective concentration of PVP to use and confirmed that 1% PVP is the most effective concentration for this process. The aim of collecting the supernatant from centrifuged lysis solution is to remove oil and proteins. The supernatant should be collected carefully to avoid retaining an oil layer in the supernatant and the pellet in the tube. These two simple modifications drastically improved the recovery, A260/A280 ratios, and A260/A230 ratios of the isolated RNA from seeds at the middle and late phases of development (Table 1).
Our method, which represents a simple modification of the standard protocol, uses commercially available kits for RNA isolation. Thus, our method is practical for isolating highly purified RNA from seeds without the need for cumbersome additional steps such as phenol-chloroform extraction and ethanol precipitation using lithium chloride. Our method is also effective for use with mature rapeseed and soybean seeds (Table 2), indicating that the modifications work well for oil- and protein-rich crop seeds. The RNA recovery rates from 4 DAF seeds (Table 1) and young roots, leaves and stems were lower using our method, indicating that our method is not suitable for RNA isolation from tissues that are poor in oil and proteins. Therefore, the method is advantageous only for the isolation of RNA from tissues containing oil, proteins, and polyphenols, such as seeds and fruits.
Overall, our method is suitable for acquiring highly purified RNA from plant seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method will facilitate more precise profiling of gene expression in seeds.

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 investigated1-6. 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 stages1,6,7. 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.

<table><tbody><tr><td>RNeasy Plant Mini Kit</td><td>QIAGEN</td><td>74904</td><td></td></tr><tr><td>polyvinylpyrrolidone</td><td>Sigma-Aldrich</td><td>P5288-100G</td><td></td></tr><tr><td>HOMOGENIZER S-303</td><td>AS ONE</td><td>1-1133-02</td><td></td></tr><tr><td>NanoDrop Lite</td><td>Thermo Scientific</td><td>ND-NDL-US-CAN</td><td></td></tr><tr><td>PrimeScript RT reagent Kit (Perfect Real Time)</td><td>TAKARA</td><td>RR037A</td><td></td></tr><tr><td>KAPA SYBR Fast qPCR kit</td><td>Kapa biosystems</td><td>KK4601</td><td></td></tr></tbody></table>

an efficient method for the isolation of highly purified rna from seeds for use in quantitative transcriptome analysis

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.

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).
<img alt="Figure 1" src="/files/ftp_upload/55008/55008fig1.jpg" /> 
Figure 1: Estimation of the optimal polyvinylpyrrolidone concentration in the cell lysis buffer. Total RNA was extracted with the cell lysis buffer containing different concentrations of PVP. (A) A photograph of the seeds after homogenization and centrifugation. (B) The graphs of wavelength absorbance for each sample are indicated. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The quantities and purity of the isolated RNAs were estimated from the graph of wavelength absorbance (Figure 1B), which showed that 1.0% PVP was the most effective concentration of isolating large amounts of purified RNA from seeds.
Developing seeds were isolated from Arabidopsis thaliana fruits on pre-chilled aluminum plates on ice under a stereomicroscope (Figures 2A, 2B). The seeds were homogenized with a stainless steel pestle and motor-grinder in a tube on an aluminum rack on ice (Figures 2C, 2D).
<img alt="Figure 2" src="/files/ftp_upload/55008/55008fig2.jpg" /> 
Figure 2: Isolation and homogenization of developing seeds of Arabidopsis thaliana. Developing seeds are isolated from fruits on a cold aluminum plate under a stereomicroscope (A). Carpels are peeled from the pedicle (B), and developing seeds are collected. The seeds are homogenized in lysis buffer containing 1% (w/v) polyvinylpyrrolidone using a stainless steel pestle (C) in tubes in an aluminum rack chilled on ice (D). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Total RNA was isolated from the seeds at 4, 8, and 12 days after flowering (hereafter referred to as DAF) using our newly developed method or the conventional method. The concentrations, A260/A280 ratios, and A260/A230 ratios of RNA isolated from 200 seeds are shown in Table 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Method</td>
			<td>DAF</td>
			<td>RNA concentration</td>
			<td>A260/A280</td>
			<td>A260/A230</td>
		</tr>
		<tr>
			<td rowspan="3">Conventional&#160;</td>
			<td>4</td>
			<td>72.4 &#177; 7.4</td>
			<td>1.90 &#177; 0.02</td>
			<td>2.27 &#177; 0.06</td>
		</tr>
		<tr>
			<td>8</td>
			<td>10.8 &#177; 3.0</td>
			<td>1.50 &#177; 0.08</td>
			<td>1.40 &#177; 0.17</td>
		</tr>
		<tr>
			<td>12</td>
			<td>&#160;4.9 &#177; 1.7</td>
			<td>1.57 &#177; 0.10</td>
			<td>0.76 &#177; 0.18</td>
		</tr>
		<tr>
			<td rowspan="3">New</td>
			<td>4</td>
			<td>63.7 &#177; 2.6</td>
			<td>2.10 &#177; 0.08</td>
			<td>2.29 &#177; 0.03</td>
		</tr>
		<tr>
			<td>8</td>
			<td>46.3 &#177; 4.9</td>
			<td>2.15 &#177; 0.05</td>
			<td>2.23 &#177; 0.09</td>
		</tr>
		<tr>
			<td>12</td>
			<td>41.7 &#177; 3.1</td>
			<td>2.09 &#177; 0.04</td>
			<td>2.17 &#177; 0.07</td>
		</tr>
	</tbody>
</table>
Table 1: Concentrations, A260/A280 ratios, and A260/A230 ratios of RNA isolated from Arabidopsis seeds. Total RNA was isolated from approximately 200 seeds (from 190 to 206 seeds) at 4, 8, and 12 DAF using the conventional method or our newly developed method. The RNA concentrations, A260/A280 ratios, and A260/A230 ratios were measured. Values represent mean &#177; SD of three independent experiments.
The results show that our method enabled us to isolate highly purified RNA from 8 and 12 DAF seeds. The total RNA was reverse-transcribed to cDNA, and the cDNA solutions were diluted 100-fold. The copy numbers of WRI1 (WRINKLED 1: AT3G54320), SDP1 (SUGAR DEPENDENT1: AT5G04040)7, and EIF3K (eukaryotic translation initiation factor 3K: AT4G33250) as an internal control8 were measured, and the relative amounts of the transcripts were estimated by quantitative real-time PCR analyses (Figure 3).
<img alt="Figure 3" src="/files/ftp_upload/55008/55008fig3.jpg" /> 
Figure 3: Quantification of low level transcripts in developing seeds. Total RNA was extracted from 4, 8, and 12 DAF seeds using our method, and cDNA solutions were prepared. The relative amounts of WRI1 (A) and SDP1 (B) transcripts were measured by quantitative real-time PCR. EIF3K was used as an internal control. Values represent mean SD of three independent experiments. DAF; days after flowering. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although the amounts of WRI1 and SDP1 transcripts are relatively low in developing seeds, it was possible to detect expression changes between seeds at different developmental stages. To verify that our method can be utilized in other oilseeds, total RNA was isolated from mature seeds of Brassica napus and Glycine max. The RNA concentrations, A260/A280 ratios, and A260/A230 ratios are shown in Table 2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Method</td>
			<td>Plant</td>
			<td>RNA concentration</td>
			<td>A260/A280</td>
			<td>A260/A230</td>
		</tr>
		<tr>
			<td>Conventional&#160;</td>
			<td>B. napus</td>
			<td>&#160;7.7 &#177; 0.8</td>
			<td>1.5 &#177; 0.12</td>
			<td>0.75 &#177; 0.11</td>
		</tr>
		<tr>
			<td></td>
			<td>G. max</td>
			<td>&#160;9.7 &#177; 2.3</td>
			<td>1.61 &#177; 0.06</td>
			<td>0.69 &#177; 0.09</td>
		</tr>
		<tr>
			<td>New&#160;</td>
			<td>B. napus</td>
			<td>143.2 &#177; 19.0</td>
			<td>2.17 &#177; 0.03</td>
			<td>2.23 &#177; 0.13</td>
		</tr>
		<tr>
			<td></td>
			<td>G. max</td>
			<td>152.3 &#177;&#160; 4.4</td>
			<td>2.17 &#177; 0.02</td>
			<td>2.30 &#177; 0.02</td>
		</tr>
	</tbody>
</table>
Table 2: Concentrations, A260/A280 ratios, and A260/A230 ratios of RNA isolated from mature seeds of Brassica napus and Glycine max. Total RNA was isolated from approximately 100 mg of Brassica napus and Glycine max seeds using the conventional method or our newly developed method. The seeds were previously crushed and roughly homogenized with a mortar and pestle chilled in liquid nitrogen. The RNA concentrations, A260/A280 ratios, and A260/A230 ratios were measured. Values represent mean &#177; SD of three independent experiments.

Watch this Scientific Journal Video about An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis at JoVE.com

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

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.

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

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9.6K Views.  National Institute for Basic Biology. 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.

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

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Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

Biology

Polysome Purification from Soybean Symbiotic Nodules

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

This protocol describes a method for eukaryotic polysome purification from intact soybean nodules. After sequencing, standard pipelines for gene expression analysis can be used to identify differentially expressed genes at the transcriptome and translatome levels.

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

Developmental Biology

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

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

Summary

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Chapters in this video

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Isolation of High Purity Tissues from Developing Barley Seeds

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Polysome Purification from Soybean Symbiotic Nodules

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples