The authors acknowledge funding from the U.S. Department of Health and Human Services, the National Institutes of Health, the National Institute of Nursing Research, and the Division of Intramural Research to Wendy A. Henderson, 1ZIANR000018-01-06; the Intramural Research Training Awards to Nicolaas H. Fourie, Ralph M. Peace, Sarah K. Abey, and Leeanne B. Sherwin; and special support from the Burroughs Wellcome Fund to Howard Hughes-NIH Research Scholar Ralph M. Peace.
The opinions expressed herein and the interpretation and reporting of these data are the responsibility of the author(s) and should not be seen as an official recommendation, interpretation, or policy of the National Institutes of Health or the United States Government.

The research described here was approved by the Institutional Review Board of the National Institutes of Health (Clinicaltrial.gov # NCT00824941).
1. Collection of Whole Blood Samples
<ol>
	<li>Collect whole blood samples (2.5 ml) from study participants via venipuncture in blood collection tubes containing an RNA stabilizing solution (allowing for long-term storage), and store them at -80 &#176;C until RNA purification.</li>
</ol>
2. Total RNA Purification
<ol>
	<li>Thaw and incubate the blood tubes for 2 hr, and then centrifuge them using a swing-out rotor for 10 min at 5,000 x g. Remove the supernatant by carefully pouring or pipetting it off into a waste tube, being careful not to disturb the pellet.</li>
	<li>Wash the pellet by adding 4 ml of RNase-free water, replace the cap securely, and vortex until the pellet is visibly dissolved. Centrifuge using a swing-bucket rotor at 5,000 x g for 10 min and remove the supernatant, as described in step 2.1.</li>
	<li>Add 350 &#181;l of BM1 buffer (provided) to the pellet. Vortex the pellet until it is visibly dissolved and transfer it to a 2-ml processing tube for automated total RNA extraction.</li>
	<li>Perform automated purification of intracellular RNA, including miRNA, from the whole blood using a commercially available RNA extraction kit, which can be automated on a robotic RNA extraction system. 
	NOTE: The automated robotic system we used can process twelve samples at a time, and it takes 3 hr for the RNA purification protocol to complete.
	<ol>
		<li>Load the pre-loaded pipette tips, centrifuge tubes, buffers, and reagents provided in the RNA purification kit into the robotic RNA extraction system.</li>
		<li>Select the &#34;Blood miRNA Part A&#34; protocol from the protocol selection menu and start the run. 
		NOTE: See Supplementary Materials for a description of the automated procedures.</li>
		<li>Once the robot has completed Part A of the automated miRNA purification protocol, empty the waste receptacle and remove the used plastics and reagent containers from the robotic system.</li>
		<li>Close the lids on all the micro-centrifuge tubes containing eluted RNA and place them in the shaker adaptor. Select &#34;Blood miRNA Part B&#34; from the protocol selection menu to incubate the samples at 65 oC for 5 min.</li>
		<li>Once the robot has completed running Part B of the miRNA purification protocol, immediately remove the samples and chill them on ice.</li>
	</ol>
	</li>
	<li>Quantitate and evaluate the purified RNA quality using a spectrophotometer. 
	NOTE: As per the protocol recommendations for the miRNA assay, a minimum concentration of 33 ng/&#181;l is required, and all samples should meet or exceed a 280/260 ratio of 1.9 and a minimum 260/230 ratio of 1.8 to ensure the absence of significant organic contamination, which can affect assay performance (refer to Reference 17).</li>
	<li>Store the samples at -80 oC.</li>
</ol>
3. miRNA Sample Preparation
<ol>
	<li>Normalize 12 RNA samples to 33 ng/&#181;l using nuclease-free water.</li>
	<li>Prepare a 1:500 dilution of the miRNA controls provided in the assay kit using nuclease-free water. Keep on ice.</li>
	<li>Prepare the annealing master mix: combine 13 &#181;l of annealing buffer (provided), 26 &#181;l of miRtag reagent (provided), and 6.5 &#181;l of miRNA controls (prepared in step 3.2) using 10- and 20-&#181;l pipettes.</li>
	<li>Using a 10-&#181;l pipette, add 3.5 &#181;l of the annealing master mix and 3 &#181;l of the RNA sample (i.e., 100 ng) to each of twelve 0.2-ml strip tubes. Flick to mix, spin down using a benchtop mini microcentrifuge (2,000 x g), and place in the thermocycler (94 oC for 1 min, 65 oC for 2 min, and 45 oC for 10 min; hold at 48 oC).</li>
	<li>Prepare the ligation master mix by combining 3 &#181;l of the ligation buffer (provided) and 19.5 &#181;l of the Polyethylene glycol (PEG; provided) using a 20-&#181;l pipette. Add 2.5 &#181;l of the ligation master mix to each of the strip tubes from step 3.4 using a 10-&#181;l pipette.
	<ol>
		<li>Mix gently, spin down using a benchtop mini microcentrifuge (2,000 x g), and return to the thermocycler at 48 oC for 5 min. After 5 min, using a 10-&#181;l pipette, add 1 &#181;l of ligase directly to each tube in the thermocycler and incubate them at 48 oC for 3 min, 47 oC for 3 min, 46 oC for 3 min, 45 oC for 5 min, and 65 oC 10 min; hold at 4 oC.</li>
	</ol>
	</li>
	<li>Remove the tubes from the thermocycler, mix them gently, spin them down using a benchtop mini microcentrifuge (2,000 x g), and add 1 &#181;l of ligation clean-up enzyme (provided). Incubate the tubes in the thermocycler (37 oC for 1 hr and 70 oC for 10 min; hold at 4 oC). Use a 10-&#181;l pipette.</li>
	<li>Remove the tubes and, using a 100-&#181;l pipette, add 40 &#181;l of nuclease-free water, mix, and spin down using a benchtop mini microcentrifuge (2,000 x g).</li>
</ol>
4. miRNA Hybridization
<ol>
	<li>Thaw the reporter and capture probe sets (provided) on ice and spin them down using a benchtop mini microcentrifuge (2,000 x g).</li>
	<li>Using a 200-&#181;l pipette, add 130 &#181;l of hybridization buffer to the reporter code set tube (130 &#181;l) to create the hybridization master mix. Mix and spin down using a benchtop mini microcentrifuge (2,000 x g). 
	NOTE: Reporter and captures probes are specific and standard for each miRNA included in the panel. Custom panels and user-designed capture and probe sets can be designed.</li>
	<li>In 12 new 0.2-ml strip tubes, add 20 &#181;l of the hybridization master mix using a 20-&#181;l pipette.</li>
	<li>Denature the miRNA samples from step 3.7 at 85 oC for 5 min and cool them on ice. Add 5 &#181;l to each of the tubes from step 4.3 using a 10-&#181;l pipette.</li>
	<li>Program the thermocycler to 65 oC at a 30-&#181;l volume-calculated temperature and heated lid at the forever time setting (do not program it to ramp down to 4 oC at the end of the run).</li>
	<li>Using a 10-&#181;l pipette, add 5 &#181;l of the capture probe set to each tube, mix, spin down using a benchtop mini microcentrifuge (2,000 x g), and immediately place in the thermocycler at 65 oC. Incubate for no less than 12 hr and no more than 30 hr.</li>
</ol>
5. Prep Station and Digital Analyzer
<ol>
	<li>Load the prep station.
	<ol>
		<li>Open the station door and load the reagent plates (provided), cartridge (provided), pipette tips (provided), prepared samples in twelve 0.2-ml strip tubes, and twelve empty 0.2-ml strip tubes (provided). Remove the strip tube caps and reagent plate cover. Close the prep-station door and run the appropriate assay from the control panel. 
		NOTE: Post-hybridization processing is the same, regardless of the assay being run. All reagents and plastic wares are prepackaged and require no preparation, other than bringing them to room temperature and spinning down the reagent-containing 96-well plates at 2,000 x g for 2 min. All components are loaded in clearly defined positions in the prep station. See Supplementary Materials for a description of the automated procedures.</li>
	</ol>
	</li>
	<li>Visualize and count the barcodes of the immobilized and oriented tripartite complexes.
	<ol>
		<li>Remove the cartridge from the prep station, seal it with the cover slips provided, and place it in the digital analyzer in the slot above the camera optic.</li>
		<li>Select the appropriate assay (miRNA panel assay) and assay version indicated on the assay kit and run the program. 
		NOTE: The digital analyzer then takes field of view images for each sample position at four excitation wavelengths, allowing the fluorescent barcodes to be read and decoded. Specific barcodes, corresponding to a specific miRNA, are counted.</li>
		<li>Export the data files to a USB or directly to the server via an internet link.</li>
	</ol>
	</li>
</ol>
6. Data Processing and Analysis
<ol>
	<li>Click on the &#34;Raw Data&#34; tab and select &#34;Import RCC Files&#34;. Select the folder containing RCC files (raw data files) from the USB.</li>
	<li>Click &#34;Next&#34;, select all QC boxes, and click &#34;Import&#34;.</li>
	<li>Click on the &#34;New Study&#34; tab. Name and describe the study and save.</li>
	<li>In the new study, select the &#34;New Experiment&#34; tab and name and describe the new experiment. Click &#34;Next&#34; and select the RLF folder from the left pane that contains the relevant raw data files that have been uploaded. Once it has been selected, click on &#34;Keep Selected&#34;, and then click &#34;Next&#34;.</li>
	<li>Add annotations if desired and click &#34;Next&#34;.</li>
	<li>Select the background subtraction method; either negative control, blank lane subtraction, or a user-defined subtraction can be selected. Once selected, click &#34;Next&#34;. 
	NOTE: Determine the most appropriate background correction approach based on the technical parameters and biological context of the study. 
	NOTE: Counts are less precise for low-abundance targets. For the purposes of the study we describe here, the data was first processed without background correction in order to examine QC parameters and to examine variation in counts for miRNA of different abundances across technical replicates. The data was then reprocessed using a 25-count background threshold.</li>
	<li>Select the &#34;Top 100&#34; normalization, and then click &#34;Next&#34;.</li>
	<li>To obtain the ratio data, select the ratio parameters, and then click &#34;Next&#34;.</li>
	<li>Click &#34;Finished&#34;.</li>
	<li>Click on the named experiment in left pane to reveal a drop down menu containing &#34;Raw&#34;, &#34;Normalized&#34;, &#34;Grouped&#34;, &#34;Ratio Data&#34;, and &#34;Analysis Data&#34;.</li>
	<li>Click on &#34;Raw Data&#34; and observe a QC report in the right pane. Observe a flag next to the samples that do not pass the default QC parameters. Build a new &#34;Study&#34; which does not include the flagged samples and reprocess as described, or simply exclude flagged QC samples from the data analysis step by selecting &#34;Exclude samples by QC/normalization flag&#34;.
	<ol>
		<li>Export &#34;Raw&#34;, &#34;Normalized&#34;, or &#34;Grouped&#34; data files in CVS or another compatible file format for analysis in another statistical or microarray software.</li>
	</ol>
	</li>
	<li>Select &#34;Normalized Data&#34; from the left pane and select the samples to be included in the analysis in the right pane.</li>
	<li>Click on &#34;Analysis&#34; and select the desired analysis type (e.g., &#34;Heat Map&#34;, &#34;Violin Plot&#34;, &#34;Box Plot&#34;, &#34;Scatter Plot&#34;, or &#34;Histogram Plot&#34;). Select the desired exclusion criteria boxes (e.g., exclude the outlier samples or genes, exclude the samples by QC flags, exclude the control genes, and/or exclude the normalization probes from data analysis).</li>
	<li>Select individual samples to be excluded from the analysis as desired.</li>
	<li>Select individual genes to be excluded from or included in the analysis as desired.</li>
	<li>Select the parameters for the selected statistical or data visualization manipulation and click on &#34;Finish&#34;.</li>
</ol>

<ol>
	<li>Aerssens, 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=Alterations+in+Mucosal+Immunity+Identified+in+the+Colon+of+Patients+With+Irritable+Bowel+Syndrome.">Alterations in Mucosal Immunity Identified in the Colon of Patients With Irritable Bowel Syndrome.</a> Clin Gastroenterol Hepatol. 6, 194-205 (2008).</li><li>Akbar, 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=Increased+capsaicin+receptor+TRPV1-expressing+sensory+fibres+in+irritable+bowel+syndrome+and+their+correlation+with+abdominal+pain.">Increased capsaicin receptor TRPV1-expressing sensory fibres in irritable bowel syndrome and their correlation with abdominal pain.</a> Gut. 57, 923-929 (2008).</li><li>Fourie, N. H., 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=Elevated+circulating+miR-150+and+miR-342-3p+in+patients+with+irritable+bowel+syndrome.">Elevated circulating miR-150 and miR-342-3p in patients with irritable bowel syndrome.</a> Exp Mol Pathol. 96, 422-425 (2014).</li><li>Zhou, Q., Souba, W. W., Croce, C. M., Verne, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-29a+Regulates+Intestinal+Membrane+Permeability+in+Patients+with+Irritable+Bowel+Syndrome.">MicroRNA-29a Regulates Intestinal Membrane Permeability in Patients with Irritable Bowel Syndrome.</a> Gut. 59, 775-784 (2010).</li><li>Zhou, Q., Verne, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNA-based+therapies+for+the+Irritable+Bowel+Syndrome.">miRNA-based therapies for the Irritable Bowel Syndrome.</a> Expert Opin Biol Ther. 11, 991-995 (2011).</li><li>Orlova, I. 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=MicroRNA+modulation+in+complex+regional+pain+syndrome.">MicroRNA modulation in complex regional pain syndrome.</a> J Transl Med. 9, 1-11 (2011).</li><li>Park, C. -. K., 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=Extracellular+MicroRNAs+Activate+Nociceptor+Neurons+to+Elicit+Pain+via+TLR7+and+TRPA1.">Extracellular MicroRNAs Activate Nociceptor Neurons to Elicit Pain via TLR7 and TRPA1.</a> Neuron. 82, 47-54 (2014).</li><li>Gheinani, A. H., Burkhard, F. C., Monastyrskaya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+microRNA+code+in+pain+and+inflammation:+lessons+from+bladder+pain+syndrome.">Deciphering microRNA code in pain and inflammation: lessons from bladder pain syndrome.</a> Cell Mol Life Sci. 70, 3773-3789 (2013).</li><li>Drossman, D. 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=.">.</a> Rome III: The Functional Gastrointestinal Disorders. , (2006).</li><li>Chey, W. D., Kurlander, J., Eswaran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irritable+bowel+syndrome:+A+clinical+review.">Irritable bowel syndrome: A clinical review.</a> JAMA. 313, 949-958 (2015).</li><li>Del Valle-Pinero, A. Y., Sherwin, L. B., Anderson, E. M., Caudle, R. M., Henderson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+vasoactive+intestinal+peptides+expression+in+irritable+bowel+syndrome+patients+and+rats+with+trinitrobenzene+sulfonic+acid-induced+colitis.">Altered vasoactive intestinal peptides expression in irritable bowel syndrome patients and rats with trinitrobenzene sulfonic acid-induced colitis.</a> World J Gastroenterol. 21, 155-163 (2015).</li><li>Henderson, W. 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=Inverse+relationship+of+interleukin-6+and+mast+cells+in+children+with+inflammatory+and+non-inflammatory+abdominal+pain+phenotypes.">Inverse relationship of interleukin-6 and mast cells in children with inflammatory and non-inflammatory abdominal pain phenotypes.</a> World J Gastrointest Pathophysiol. 3, 102-108 (2012).</li><li>Pekow, J. R., Kwon, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+inflammatory+bowel+disease.">MicroRNAs in inflammatory bowel disease.</a> Inflamm Bowel Dis. 18, 187-193 (2012).</li><li>Veldman-Jones, M. H., 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=Flexible+Molecular+Subtyping+of+Clinical+DLBCL+Samples+Using+the+NanoString+nCounter+System.">Flexible Molecular Subtyping of Clinical DLBCL Samples Using the NanoString nCounter System.</a> Clin Cancer Res. 21, 2367-2378 (2015).</li><li>Lee, 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=Nanostring-Based+Multigene+Assay+to+Predict+Recurrence+for+Gastric+Cancer+Patients+after+Surgery.">Nanostring-Based Multigene Assay to Predict Recurrence for Gastric Cancer Patients after Surgery.</a> PLoS ONE. 9, e90133 (2014).</li><li>Lohavanichbutr, P., 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=A+13-gene+signature+prognostic+of+HPV-negative+OSCC:+discovery+and+external+validation.">A 13-gene signature prognostic of HPV-negative OSCC: discovery and external validation.</a> Clin Cancer Res. 19, 1197-1203 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> nCountermiRNA Expression Assay User Manual (MAN-C0009-05). , (2013).</li></ol>

W.A.H., S.K.A, N.H.F., and R.M.P. hold a US patent (no. PCT/US14/38638) for a circulating miRNA signature of IBS emanating from the research described herein.

Critical Steps within the Protocol
For the miRNA assay in particular, it is critical not to use unpurified lysates (these can be used in mRNA and protein assays), as the reagents have not been optimized for use with lysates, and sample preparation reactions are likely to be inhibited. Additionally, contaminants carried over from lysis and RNA purification steps (e.g., guanidinium compounds, ethanol, and phenols) can inhibit ligation and sample purification reactions. Good-quality RNA is therefore critical to the sensitivity and accuracy of the miRNA assay.
Using a thermocycler with a heated lid is critical to ensure proper temperature control to optimize the performance of all reaction steps. During step 3.5.1, it is important not to remove the strips from the thermocycler in order to maintain the temperature during the addition of the ligase. Removing the strips to add the ligase will compromise the reaction. When performing the hybridization steps, it is critical to avoid vigorous shaking, pipetting, or flicking when mixing reagents in the tubes, as this may shear the reporter probes. To ensure optimal performance and consistency, it is important to thoroughly mix reagents in the tubes by gentle flicking. Always spin down reactions after mixing, and use a new pipette tip each time a reagent is dispensed to ensure accurate pipetting and to avoid accidental cross-contamination.
Modifications and Troubleshooting
The code sets can be customized to include only targets of interest. In this way, costs can be balanced to allow for the testing of large sample sets when further investigating or validating a bio-signature. Custom probes can be designed for genes or targets not available from the a la carte menu. Sensitivity for less-abundant targets or low-concentration RNA can be manipulated by allowing for a longer hybridization time and/or by using higher-resolution digital counting.
In our study we used the predefined 800 target miRNA panel with total RNA from whole blood; however, the assays can be customized to include fewer miRNAs if a more targeted approach is needed. A total of 24 samples can be prepared on a single day, staggered in two sets of 12, because two cartridges can comfortably be passed through post-hybridization processing on the robotic prep station and imaged on the digital analyzer the next day. Staggered processing of the samples in batches of 12 is important to standardize the hybridization time. Cartridges that have been imaged can be saved and stored at 4 oC to be rescanned if data analyses suggest that a higher resolution scan may improve the detection of rarer miRNAs. Detection of rarer molecules may also be improved by hybridizing samples for longer; however, prolonged hybridization may result in oversaturation and may only improve results if the starting RNA concentrations are low (as in extracellular vesicle-derived RNA). The platform&#39;s sensitivity and precision allow relatively low-abundance miRNAs to be reliably counted.
Limitations of the Technique
The described gene expression assay platform only accommodates up to 800 targets per array. It is therefore not suited for whole miRNA-transcriptome/whole transcriptome studies. Only known, described, and specified targets can be detected using the platform, so it is not suited to the discovery of new molecular species. Other methods, such as RNASeq or other traditional microarray methods, are more suited to whole transcriptome exploratory studies.
Significance of the Technique with Respect to Existing/Alternative Methods
IBS is characterized by a combination of symptoms, principally including abdominal pain; visceral hypersensitivity; and changes in bowel habits, such as frequent diarrhea, constipation, or a combination of both9,10. IBS symptoms have no known organic cause, and patients do not present with clinically significant inflammation, histopathological perturbations of gastrointestinal tissues, or systemic markers9-12. Research suggests that biological dysregulation in IBS is subtle, subclinical, and heterogeneous, with common themes emerging around inflammation and immune function10. Therefore, we needed to control experimenter-introduced variation and use a platform that was capable of reliably detecting subtle perturbations in gene expression. The unique attributes of the assay and system standardize sample processing and reduce experimenter handling through automation, reducing technical variance to produce highly reproducible data. These features allow for focused investigations and produce highly precise and replicable data. This allows for the detection of subtle perturbations and perturbations among low-expression targets that may be overlooked or swamped by the signals of more abundant targets when using intensity- or amplification-based methods. PCR-based microarrays and RNAseq methods are viable alternatives to using the described platform and may be attractive as alternative when higher throughputs are required or when the aim is to characterize the whole transcriptome or to look for novel molecules. However, alternatives may not perform as well in accurately and sensitively detecting and quantitating rare targets and subtle perturbations.
Future Applications or Directions after Mastering this Technique
Circulating miRNA expression in IBS participants showed a number of perturbations that were found to be both significant and consistent within categories. The identified miRNAs were associated with relevant pathways and pathological conditions, including a functional pain condition (miR-342-3p: chronic bladder pain)8 and inflammation (miR-150)13. The example study was based on an exploratory cohort used to define targets and systems of interest. A more targeted, smaller miRNA array was then constructed, lowering the per sample cost and allowing for much larger cohorts to be analyzed in a cost-effective manner in order to validate and refine signatures and biomarkers. The gene expression platform assay system strikes a balance between the traditional exploratory nature of the microarray and more targeted, hypothesis-driven data collection to refine and test molecular signatures and biomarkers14-16. The method will be used to examine molecular and protein perturbations in in vivo and in vitro models where the described target miRNAs are experimentally over-expressed or inhibited. New assays allow for the simultaneous quantification of mRNAs and proteins directly from cell and tissue lysates. Furthermore, by optimizing the purification of specific fractions of peripheral blood and excreta (e.g., urine) and by customizing and optimizing certain assay parameters, molecular profiles and signatures of low molecular concentration fractions (e.g., exosomal fractions) will be examined.

Irritable Bowel Syndrome (IBS) is the most common outpatient diagnosis in Gastroenterology, afflicting 10 - 15% of the general population and representing a significant burden on health services. Currently, there are no generally accepted diagnostic biomarkers for IBS (i.e., the diagnosis is based on clinical symptoms and ruling out other organic disorders, such as GI malignancy, Inflammatory Bowel Diseases, and gastrointestinal (GI) infections). When studying gene expression profiles in common conditions with subclinical histopathology, such as IBS, high sensitivity and accuracy (reproducibility) are needed, as perturbations in gene expression profiles are often subtle1-3. miRNAs have been identified as both diagnostic and functional targets in IBS4,5, and we are particularly interested in assessing their use as circulating biomarkers of IBS-associated biological perturbations3,4,6,7. The clinical utilization of circulating biomarkers is of current interest due to the ease and minimally invasive nature of the collection of the biomarkers&#39; source (e.g., peripheral blood) from patients.
Gene expression platform assays, because of their unique chemistry and streamlined, mostly automated workflow, provide a microarray platform that provides wide and customizable coverage (800 targets in a single reaction) of the transcriptome for targeted discovery. They also yield high precision and linearity over a wide spectrum of expression levels in the quantification of transcriptomic targets. The assay does not require the reverse transcription of RNA, the amplification of resulting cDNA, or any other enzymatic reactions. Thus, potential errors introduced by the high cycle numbers of amplification from RNA species with low abundance or rare splice variants are reduced by the process of digital counting. This means that a wide variety of sample types, such as purified total RNA (i.e., mRNA + miRNA), RNA from formalin-fixed paraffin-embedded (FFPE) specimens, as well as crude tissue and cell lysates, can be used with the assays.
RNAs of interest are detected using a pair of probes (capture and reporter probes), each containing a target-specific sequence that recognizes a corresponding region on the target RNA. In order to ensure the detection of short RNAs (i.e., miRNAs), the mature miRNA sequence is elongated by annealing a unique oligonucleotide tag (miRtag) to the 3&#39; end of the molecule. A bridging oligonucleotide, which is complimentary to a portion of both the mature target miRNA and the miRNA-specific miRtag, is used to ensure sequence specificity. Capture and reporter probes ligate to the 3&#39; and 5&#39; ends of the mature miRNA-miRtag complex, respectively. The capture probes have a biotin label at the 3&#39; end, which allow them to attach to the surface of a streptavidin-coated glass slide, fixing the capture probe-miRNA-miRtag-reporter probe complex to the slide. An electric current is then used to orient the complex on the slide surface, allowing fluorescent barcodes carried by the reporter probe on the 5&#39; end to be visualized using a microscope and charge-coupled device (CCD) camera. Barcodes are counted and decoded, yielding a count of the specific target miRNAs. The fluorescent barcode consists of six positions that can be occupied by one of four fluorescent colors, which can be used to construct over 4,000 color-barcode combinations, each coding for a specific RNA.
Sample preparation (i.e., RNA purification or cell/tissue lysate preparation), as well as hybridization of the capture and reporter probes, are done at the bench. Twelve specimens can be multiplexed on a single slide. After the overnight hybridization, all further specimen preparation is performed by a robotic prep station. The loaded slides are then transferred to a digital analyzer for imaging, counting, decoding, and data processing. The resulting data are imported to the accompanying software, where they are further processed with a high degree of user control. The unique chemistry, simple preparation, automated nature, simple data type (counts), and overall streamlined workflow of the assay facilitate high-precision gene expression quantification, making it a useful tool in studying circulating miRNA expression profiles and signatures in functional conditions such as IBS.

<table border="0" cellpadding="0" cellspacing="0" width="758">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:221px;">nCounter Prep-Station</td>
			<td style="width:221px;">Nanostring</td>
			<td style="width:108px;">N/A</td>
			<td style="width:208px;">Essential</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">nCounter Digital Analyzer</td>
			<td>Nanostring</td>
			<td>N/A</td>
			<td>Essential</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectrophotometer</td>
			<td>NanoDrop</td>
			<td>ND-1000</td>
			<td>Can use suitable equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Can use suitable equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MiniMicroCentrifuge</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Can use suitable equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipettes (1000, 100, 20, 10ul)</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Can use suitable equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qiacube</td>
			<td>Qiagen</td>
			<td>9001292</td>
			<td>Can use suitable equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PAXgene Blood miRNA Kit</td>
			<td>Qiagen</td>
			<td>763134</td>
			<td>Can use suitable equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PAXgene Blood Tubes</td>
			<td>VWR</td>
			<td>77776-026</td>
			<td>Can use suitable equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">nCounter Human miRNA Assay Kit</td>
			<td>Nanostring</td>
			<td>150625</td>
			<td>Essential</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">nCounter Master Kit</td>
			<td>Nanostring</td>
			<td>100050</td>
			<td>Essential</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">nSolver</td>
			<td>Nanostring</td>
			<td>N/A</td>
			<td>Essential</td>
		</tr>
	</tbody>
</table>

perturbations of circulating mirnas in irritable bowel syndrome detected using a multiplexed high-throughput gene expression platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

Transcriptomic perturbation in functional disorders may be subtle, and in order to reliably detect those subtle changes in expression, quantification of gene expression needs to be precise. The gene expression platform assay system reduces technical noise through its highly automated nature, and the unique chemistry it uses produces high-precision gene expression data. Technical replicates shown in Figure 1 demonstrate the high reproducibility in both endogenous (blue dots) and viral miRNA (orange dots; housekeeping genes are represented by yellow dots) expression quantification that is possible using this method. Using this method, viral (Figure 2a) and endogenous (Figure 2b) miRNAs that show deviation from expected expression, as defined by healthy study participants, can be identified as targets of interest in IBS. It should be noted that it is unclear whether the viral miRNAs detected here derive from viral genes integrated into the human genome or from viral load. As the assay only counts mature miRNAs with single-nucleotide specificity, cross hybridization with similar endogenous human miRNAs is unlikely. Nonetheless, differential steady-state levels of these molecules are of interest as biomarkers. The precision of the method allows for perturbations among low expression targets to be detected, as accurate detection of these transcripts are not swamped out by more abundant transcripts. Subtle perturbations can also be reliably observed. Figures 3 and 4 show some of these perturbations in circulating miRNAs in IBS and IBS-subtypes compared to healthy controls, and Figures 5 and 6 (both adapted from 3) show the two most significant differentially elevated miRNAs (Figure 5: miRNA 342-3p, Figure 6: miRNA 150) in IBS participants. Figure 6 is an example of the graphical output of results generated by the associated software.
<img alt="Figure 1" src="/files/ftp_upload/54693/54693fig1.jpg" /> 
Figure 1: Normalized Counts from two Technical Replicates. Technical replicates run on two separate cartridges show high linear correlation (r2 = 0.90, y = 1.03x - 0.4) over the range of the normalized (normalized to the top 100 expressed miRNAs) miRNA counts (log2 counts on the x- and y-axis). The scatter plot includes data for endogenous human miRNAs in blue (654 miRNAs), endogenous viral miRNAs in orange (80 miRNAs), and housekeeping genes in yellow (8 genes). Endogenous viral miRNAs were available on earlier versions of the assay (version 1.4 was used here), but are no longer included on the standard assay, although they are available as custom additions. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54693/54693fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54693/54693fig2.jpg" /> 
Figure 2: Normalized Counts of Viral and Endogenous Human miRNAs in IBS-constipation (IBS-C) versus Healthy Controls. Perturbations in (a) viral (orange) and (b) endogenous human miRNAs (blue) are evident in this example, where miRNA counts (log2 transformed) of IBS-C participants (y-axis) are regressed against the miRNA counts (log2 transformed) of healthy controls (x-axis). The majority of miRNAs express very similarly in both populations, but some notably deviate from the correlation. These deviations are evident among both rare and abundantly expressed targets. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54693/54693fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54693/54693fig3.jpg" /> 
Figure 3: miRNAs are Significantly and Uniformly Differentially Expressed in IBS Compared to Healthy Controls. Differentially expressed miRNAs in IBS are revealed using the linear discriminant analysis effect size method, which combines tests of statistical significance with checks for consistency within categories. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54693/54693fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54693/54693fig4.jpg" /> 
Figure 4: miRNAs are Significantly and Uniformly Differentially Expressed in IBS Subtypes Compared to Healthy Controls. miRNAs were differentially expressed in IBS-subtypes (-D: diarrhea, -C: constipation) compared to healthy controls using the linear discriminant analysis effect size method, which combines tests of statistical significance with checks for consistency within categories. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54693/54693fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54693/54693fig5.jpg" /> 
Figure 5: Differential Expression of miRNA-342-3p. Chronic abdominal pain of unknown etiology is a principal symptom of IBS. The box plot shows that miR-342-3p (normalized counts on the y-axis), a miRNA associated with chronic bladder pain of unknown etiology, was found to be present in circulation at higher counts across all IBS subtypes compared to healthy controls. The bars represent the non-outlier range, the boxes the inter-quartile range and the blue square the median count. Modified from 3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54693/54693fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54693/54693fig6.jpg" /> 
Figure 6: Differential Expression of miRNA-150. A violin plot showing that IBS participants had significantly higher circulating miR-150 counts compared to healthy controls (log2 transformed counts on the y-axis). The curves indicate the frequency of counts in the category, the vertical bars represent the 1st and 3rd quartiles, and the red square indicates the median count. Modified from 3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54693/54693fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform at JoVE.com

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

perturbations-circulating-mirnas-irritable-bowel-syndrome-detected

Research

JoVE Journal

Genetics

8.2K Views.  National Institutes of Health, DHHS. We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome. 

Video: Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...