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

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

1. 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 °C until RNA purification.

2. Total RNA Purification

1. 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.
2. 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.
3. Add 350 µl 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.
4. 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.
   1. Load the pre-loaded pipette tips, centrifuge tubes, buffers, and reagents provided in the RNA purification kit into the robotic RNA extraction system.
   2. Select the "Blood miRNA Part A" protocol from the protocol selection menu and start the run.
      NOTE: See Supplementary Materials for a description of the automated procedures.
   3. 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.
   4. Close the lids on all the micro-centrifuge tubes containing eluted RNA and place them in the shaker adaptor. Select "Blood miRNA Part B" from the protocol selection menu to incubate the samples at 65 ^oC for 5 min.
   5. Once the robot has completed running Part B of the miRNA purification protocol, immediately remove the samples and chill them on ice.
5. 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/µl 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).
6. Store the samples at -80 ^oC.

3. miRNA Sample Preparation

1. Normalize 12 RNA samples to 33 ng/µl using nuclease-free water.
2. Prepare a 1:500 dilution of the miRNA controls provided in the assay kit using nuclease-free water. Keep on ice.
3. Prepare the annealing master mix: combine 13 µl of annealing buffer (provided), 26 µl of miRtag reagent (provided), and 6.5 µl of miRNA controls (prepared in step 3.2) using 10- and 20-µl pipettes.
4. Using a 10-µl pipette, add 3.5 µl of the annealing master mix and 3 µl 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).
5. Prepare the ligation master mix by combining 3 µl of the ligation buffer (provided) and 19.5 µl of the Polyethylene glycol (PEG; provided) using a 20-µl pipette. Add 2.5 µl of the ligation master mix to each of the strip tubes from step 3.4 using a 10-µl pipette.
   1. 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-µl pipette, add 1 µl 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.
6. Remove the tubes from the thermocycler, mix them gently, spin them down using a benchtop mini microcentrifuge (2,000 x g), and add 1 µl 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-µl pipette.
7. Remove the tubes and, using a 100-µl pipette, add 40 µl of nuclease-free water, mix, and spin down using a benchtop mini microcentrifuge (2,000 x g).

4. miRNA Hybridization

1. Thaw the reporter and capture probe sets (provided) on ice and spin them down using a benchtop mini microcentrifuge (2,000 x g).
2. Using a 200-µl pipette, add 130 µl of hybridization buffer to the reporter code set tube (130 µl) 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.
3. In 12 new 0.2-ml strip tubes, add 20 µl of the hybridization master mix using a 20-µl pipette.
4. Denature the miRNA samples from step 3.7 at 85 ^oC for 5 min and cool them on ice. Add 5 µl to each of the tubes from step 4.3 using a 10-µl pipette.
5. Program the thermocycler to 65 ^oC at a 30-µl 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).
6. Using a 10-µl pipette, add 5 µl 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.

5. Prep Station and Digital Analyzer

1. Load the prep station.
   1. 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.
2. Visualize and count the barcodes of the immobilized and oriented tripartite complexes.
   1. 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.
   2. 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.
   3. Export the data files to a USB or directly to the server via an internet link.

6. Data Processing and Analysis

1. Click on the "Raw Data" tab and select "Import RCC Files". Select the folder containing RCC files (raw data files) from the USB.
2. Click "Next", select all QC boxes, and click "Import".
3. Click on the "New Study" tab. Name and describe the study and save.
4. In the new study, select the "New Experiment" tab and name and describe the new experiment. Click "Next" 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 "Keep Selected", and then click "Next".
5. Add annotations if desired and click "Next".
6. Select the background subtraction method; either negative control, blank lane subtraction, or a user-defined subtraction can be selected. Once selected, click "Next".
   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.
7. Select the "Top 100" normalization, and then click "Next".
8. To obtain the ratio data, select the ratio parameters, and then click "Next".
9. Click "Finished".
10. Click on the named experiment in left pane to reveal a drop down menu containing "Raw", "Normalized", "Grouped", "Ratio Data", and "Analysis Data".
11. Click on "Raw Data" 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 "Study" which does not include the flagged samples and reprocess as described, or simply exclude flagged QC samples from the data analysis step by selecting "Exclude samples by QC/normalization flag".
    1. Export "Raw", "Normalized", or "Grouped" data files in CVS or another compatible file format for analysis in another statistical or microarray software.
12. Select "Normalized Data" from the left pane and select the samples to be included in the analysis in the right pane.
13. Click on "Analysis" and select the desired analysis type (e.g., "Heat Map", "Violin Plot", "Box Plot", "Scatter Plot", or "Histogram Plot"). 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).
14. Select individual samples to be excluded from the analysis as desired.
15. Select individual genes to be excluded from or included in the analysis as desired.
16. Select the parameters for the selected statistical or data visualization manipulation and click on "Finish".