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 border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RNeasy Plant Mini Kit</td>
			<td style="width:81px;">QIAGEN</td>
			<td style="width:136px;">74904</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">polyvinylpyrrolidone</td>
			<td style="width:81px;">Sigma-Aldrich</td>
			<td style="width:136px;">P5288-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HOMOGENIZER S-303</td>
			<td style="width:81px;">AS ONE</td>
			<td style="width:136px;">1-1133-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">NanoDrop Lite</td>
			<td style="width:81px;">Thermo Scientific</td>
			<td style="width:136px;">ND-NDL-US-CAN</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PrimeScript RT reagent Kit (Perfect Real Time)</td>
			<td style="width:81px;">TAKARA</td>
			<td style="width:136px;">RR037A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">KAPA SYBR Fast qPCR kit</td>
			<td style="width:81px;">Kapa biosystems</td>
			<td style="width:136px;">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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Nanyang Technological University

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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

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

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 ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.