Rna isolation reagent

The corneal tissue was cut into small pieces by microscissors and lysed in RNA isolation reagent (RNA Bee, Tel-Test, Inc., Friendswood, TX). After sonication with a probe sonicator (Ultrasonic Processor, Cole Parmer Instruments, Vernon Hills, IL), total RNA was extracted using an RNeasy Mini kit (Qiagen, Valencia, CA), and first-strand cDNA was synthesized by reverse transcription (High Capacity RNA-to-cDNA Kit; Applied Biosystems, Carlsbad, CA). The cDNA was analyzed by real-time PCR using TaqMan Universal PCR Master Mix (Applied Biosystems) on an ABI 7500 Real-Time PCR System (Applied Biosystems) for the following molecules: rabbit ABCG2 (ATP-binding cassette sub-family G member 2), FGF2 (fibroblast growth factor 2), interleukin (IL)-1β, and IL-6. A rabbit GAPDH was used for normalization of gene expression. For probe sets, TaqMan Gene Expression Assay kits were purchased from Applied Biosystems. The assays were performed in triple technical replicates for each sample.

For RNA extraction, the cells were lysed in RNA isolation reagent (RNA Bee, Tel-Test Inc., Friendswood, TX) and homogenized with an ultrasound sonicator (Ultrasonic Processor, Cole Parmer Instruments, Vernon Hills, IL). Total RNA was extracted using RNeasy Mini kit (Qiagen, Valencia, CA). After the amount of RNA was measured using Nanodrop 1000 spectrophotometer (Thermo scientific, Waltham, MA), 1 μg RNA was used to generate cDNA by reverse transcription (High Capacity RNA-to-cDNA Kit, Applied Biosystems, Carlsbad, CA). Real-time amplification was performed using TaqMan^® Universal PCR Master Mix (Applied Biosystems) in ABI 7500 Real Time PCR System (Applied Biosystems) for the following molecules: NLRP3, IL-1β, IL-6, IL-8, TNF-α, MCP-1, IκBα, p62/SQSTM1, Nrf2 and NQO1 (NAD(P)H dehydrogenase quinone 1). Human PCR probe sets were commercially purchased (TaqMan^® Gene Expression Assay Kits, Applied Biosystems). Values were normalized to 18s RNA and expressed as fold changes relative to controls.

For RNA extraction, cells or tissues were lysed using RNA isolation reagent (RNA Bee, Tel-Test, Friendswood, TX) and homogenized with an ultrasound sonicator (Ultrasonic Processor, Cole Parmer Instruments, Vernon Hills, IL). Total RNA was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany) and converted to first-strand cDNA by reverse transcription (High Capacity RNA-to-cDNA Kit, Applied Biosystems, Carlsbad, CA). Then, real-time amplification was carried out using TaqMan Universal PCR Master Mix (Applied Biosystems) in the ABI 7500 Real-Time PCR System (Applied Biosystems). All PCR probe sets (TaqMan Gene Expression Assay kits) were purchased from Applied Biosystems. Data were normalized to Gapdh and expressed as fold changes relative to controls.