Small RNA-seq Aqueous humor of human glaucoma (open-angle glaucoma and pseudoexfoliative glaucoma) patients against cataract
Aqueous humor samples were procured from patients slated for elective glaucoma or cataract surgeries. Intraoperative collection was performed via ab-externo limbal paracentesis using a 27-gauge needle attached to a tuberculin syringe. Rigorous precautions were instituted to prevent vascular interference and to ensure the integrity of the samples against contamination. A volume ranging from 50 to 200 microliters of aqueous humor was aspirated into a cryovial prior to commencing the primary corneal incision, after which the samples were promptly stored in dry ice within the operating theater. Specimens exhibiting evidence of hemorrhage or failing to meet the minimum volume threshold of 50 microliters were excluded from subsequent analysis. Successfully collected and preserved aqueous humor samples—immediately snap-frozen post-extraction—were transported in batches under stringently controlled conditions (maintained frozen on dry ice) to the molecular analysis laboratory. The samples of aqueous humor were thawed slowly on ice and centrifuged at 3000g for 15 minutes at 4°C to remove cells and cell debris, with the supernatant collected for the extraction of RNA. The supernatant were completed up to 200 µl volume with a 10 mM Tris-HCL pH 8.0 buffer, and RNA was extracted using a commercial kit for RNA purification (miRNeasy Serum/Plasma Advanced Kit; Qiagen, Hilden, Germany), according to the manufacturer's recommendations. The total yield of purified RNA from each patient was fully employed to generate sequencing libraries using the high-sensitivity, low-input TrueQuant Small RNA-Seq Library Preparation Kit (GenXPro GmbH, Frankfurt, Germany), adhering meticulously to the manufacturer's protocol. Briefly, small RNA molecules were ligated to 3' and 5' adapters containing TrueQuant Unique Molecular Identifiers (UMIs) (TrueQuant Adapters; GenXPro GmbH, Frankfurt, Germany), followed by reverse transcription and polymerase chain reaction (PCR) amplification with sample-specific indexed primers. Amplification was conducted with a reduced cycle number to minimize bias, succeeded by purification via magnetic bead-based separation. The resultant libraries underwent quality evaluation using a DNA analysis cartridge (Agilent High Sensitivity DNA Kit; Agilent Technologies, Santa Clara, CA, USA) on a microfluidics-based system (Agilent Bioanalyzer 2100; Agilent Technologies).