Tear-Derived Exosomal miR-15a as New Diagnostic Tool for Diabetic Retinopathy

Our research explores the potential of tear-derived exosomal microRNA-15a as a non-invasive biomarker for diabetic retinopathy, aiming to improve early diagnosis and patient outcomes in diabetic-related eye diseases. Current research leverages droplet digital PCR, exosome isolation, and molecular profiling of tear-based microRNAs to enhance diagnostic accuracy and biomarker discovery for diabetic retinopathy. Current challenges include limited tear volume, absence of specialized tear isolation kits, and lack of a validated endogenous reference microRNA, complicating normalization and consistent quantification in tear-based diagnostics.

We identify tear-derived extracellular vesicular or exosomal biomarkers as a non-invasive tool for diabetic retinopathy, though DR and non-DR groups overlap, finding support tear fluids potential for early disease detection. We will focus on how other microRNAs regulate diabetic retinopathy, explore the use as a non-invasive biomarker, and investigate their role in other diseases to prevent vision loss. To begin, take a tube containing the Schirmer strip soaked in PBS and vortex it for five minutes at room temperature.

Place the tube on a rocker and agitate it for five minutes at room temperature. Now, centrifuge the tube at 2, 000 G for 15 minutes at four degrees Celsius. Transfer the solution into a new labeled tube and store it at minus 80 degrees Celsius until further processing.

For the exosome isolation, thaw the tear solution on ice for 10 minutes. Equilibrate the spin column for 15 minutes at room temperature. Then remove the lower outlet of the column and position it over the provided waste collection plate.

Remove the top ceiling mat and allow the storage buffer to pass through the column by gravity. Then add 250 microliters of PBS and let it pass through the column. Apply 110 microliters of a sample onto the top of each column and allow it to enter the column.

Move the column onto a provided sample collection plate. Add 100 microliters of PBS to the top and allow it to pass through the column into the sample collection plate. Place the column back onto the waste collection plate.

Add 200 microliters of PBS four times to the column and allow it to enter the column to remove the free protein fraction from the first loading. Then add 110 microliters of the sample into the column and allow it to enter. Place the column onto the sample collection plate.

Add 100 microliters of PBS and allow it to pass through the column. Briefly centrifuge the sample collection plate at 100 G for 30 seconds. The isolated exosomes are now ready for RNA isolation.

Take 200 microliters of the isolated exosomes and add 60 microliters of lysis buffer to it. Vortex for five seconds and incubate the mixture at room temperature for three minutes. Add 20 microliters of inhibitor precipitation buffer.

Vortex again for 20 seconds before incubating for three minutes at room temperature. Centrifuge the tube at 12, 000 G for three minutes at room temperature. Transfer the clear colorless supernatant to a new tube and add one volume of isopropanol to it.

After vortexing for five seconds, transfer the solution to the mini spin column provided in the kit. Centrifuge the column at room temperature for 15 seconds at 8, 000 G and discard the flowthrough. Wash the column with 700 microliters of RWT wash buffer.

After centrifuging for 15 seconds, discard the flowthrough. Now wash the column with 500 microliters of RPE wash buffer before centrifuging again for 15 seconds. Discard the flowthrough and add 500 microliters of 80%ethanol to the sample mini spin column before centrifuging again.

After discarding the flowthrough and the collection tube, place the mini spin column into a new two milliliter collection tube. Centrifuge at 18, 000 G for five minutes and discard the flowthrough and collection tube. Place the mini spin column into a new 1.5 milliliter tube.

Then add 15 microliters of RNAse-free water at the center of the spin column membrane. After incubating for three minutes at room temperature, centrifuge the tube at 18, 000 G for one minute to elute the RNA. Nanoparticle tracking analysis was used to evaluate the size distribution and concentration of exosomes isolated from tear samples.

The average size of exosomes isolated from tears was approximately 142.4 nanometers. The total number of exosomes in the sample diluted to 1:20 was 3.9 times 10 to the 10th particles per milliliter. The copy number of microRNA-15a significantly increased in patients with diabetes without retinopathy compared to healthy controls, suggesting early elevation of this biomarker with diabetic onset.

A further significant increase in microRNA-15a copy number was observed in patients with diabetic retinopathy compared to controls, reinforcing its association with diabetic conditions. No statistically significant difference in microRNA-15a levels was found between the diabetes without retinopathy and diabetic retinopathy groups, indicating that while miR-15a reflects diabetic presence, it does not differentiate between stages of retinopathy.