The overall goal of this multiplex nucleic acid quantification protocol is to digitally quantify microRNA expression in whole blood. This method can help answer key questions in the study of digestive disorders. It helps with identification of dysregulated molecular mechanisms and biomarker signatures, like microRNAs.

The main advantage of this technique is that microRNA quantification does not rely on the amplification. This technology digitally counts molecular targets and is largely automated. To purify total RNA, thaw and incubate previously collected and frozen whole blood samples for two hours.

Use a swinging bucket rotor to centrifuge the tubes at 5, 000 times g at room temperature for ten minutes. Then, remove the supernatants by carefully pouring or pipetting it into a waste tube, taking care not to disturb the pellet. Add four milliliters of RNase-free water to the pellet.

Securely replace the cap, and vortex until the pellet is visibly dissolved. Then, centrifuge the tube again, and remove the supernatant. Next, add 350 microliters of BM1 buffer to the pellet and vortex until the pellet is visibly dissolved.

Then, transfer the sample to a two milliliter processing tube for automated total RNA extraction. Using a commercially-available RNA extraction kit, carry out an automated purification of intracellular RNA, including microRNA from the whole blood sample, by loading pre-loaded pipette tips, centrifuge tubes, buffers, and reagents provided in the RNA purification kit into the robotic RNA extraction system. From the protocols selection menu, choose the Blood miRNA PartA protocol, and start the run.

Complete the RNA extraction, and store the samples according to the text protocol. Prepare microRNA samples by first using RNase-free water to normalize 12 RNA samples to 33 nanograms per microliter. With nuclease-free water, prepare a 1-in-500 dilution of the microRNA controls provided in the assay kit, and keep on ice.

Next, prepare the annealing master mix by combining 13 microliters of annealing buffer, 26 microliters of micro R-tag reagent, and 6.5 microliters of microRNA controls. To each of 12 0.2 milliliters strip tubes, add 3.5 microliters of the annealing master mix, and then add 3 microliters of the RNA sample. Flick the tubes to mix, spin them down at 2000 times g with a bench-top mini microcentrifuge, and run the tubes in a thermocycler using the following program.

Next, prepare a ligation master mix by combining three microliters of the ligation buffer and 19.5 microliters of PEG. Then, add 2.5 microliters of the ligation master mix to each of the reactions. After mixing and spinning down the tubes, return them to the thermocycler at 48 degrees Celsius for five minutes.

Then add one microliter of ligase directly to each sample while still in the thermocycler block, and incubate the tubes using the following program. Gently mix and spin down the tubes, and add one microliter of ligation cleanup enzyme to each sample. Then, incubate the tubes in the thermocycler as shown here.

After the incubation, add 40 microliters of RNase-free water to the samples before mixing and spinning them down. To carry out microRNA hybridization, thaw the reporter and capture probe sets provided on ice, and spin them down. Add 130 microliters of hybridization buffer to the Reporter Code Set 2, and mix to create the hybridization master mix.

In 12 new 0.2 milliliter strip tubes, add 20 microliters of the hybridization master mix. Denature the microRNA samples just prepared at 85 degrees Celsius for five minutes, and cool them on ice. Then, add 5 microliters of the microRNA samples to each of the strip tubes containing the hybridization master mix.

Program the thermocycler to 65 degrees Celsius, at a 30 microliters volume-calculated temperature, and the heated lid at the Forever time setting so that it does not ramp down to 4 degrees Celsius at the end of the run. Add 5 microliters of the capture probe set to each tube, and mix. Then, after spinning them down, immediately place the tubes in the thermocycler at 65 degrees Celsius.

Incubate the samples for no less than 12 hours, and no more than 30 hours. Set up the prep station by first warming the cartridge and plates to room temperature. Then spin down the plates.

To load the prep station, open the station door, and load the reagent plates, cartridge, pipette tips, prepared samples in 0.2 milliliter strip tubes, and two empty 0.2 milliliter 12-tube strip tubes to the appropriate locations in the robot. Remove the strip tube caps and reagent plate cover. Then, close the prep station door, and run the appropriate assay from the control panel.

To visualize and count the barcodes of the immobilized and oriented tripartite complexes, remove the cartridge from the prep station, use the cover provided to seal it, and place it in the digital analyzer in any available slot. Use the touch screen to create a cartridge definition file, or CDF. Ensure that the appropriate reporter library file is assigned to each sample in the CDF.

Once the scanning is complete, download the data files from the digital analyzer using a USB drive or via email, before processing and analyzing the data, according to the associated QC and analysis protocol. 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. The technical replicates shown here demonstrate the high-reproducibility in both endogenous and viral microRNA expression quantification that is possible.

Using this method, viral-encoded and endogenous human microRNAs that show deviation from the expected expression, as defined by healthy study participants, can be identified as targets of interest in IBS. The precision and sensitivity of the method allows for perturbations among low-expression targets to be detected, and subtle perturbations can also be reliably observed. These figures show some of the perturbations in circulating microRNAs in IBS, and IBS subtypes, compared to healthy controls.

The two most significant differentially-elevated microRNAs in IBS participants are presented here. The curves in this violin plot indicate the frequency of counts for miR-150. The vertical bars represent the first and third quartiles and the red square indicates the median count.

Following this procedure, more targeted custom assays based on the same technology can be performed in order to validate biomarker signatures in larger cohorts and explore the essential RNA and protein associations. After its development, this technique paved the way for researchers in the field of biomedical research to rapidly, with precision and accuracy, develop diagnostic biomarker panels for disease.

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