Rneasy plus micro rna isolation kit

Murine corneal epithelium and conjunctival tissue were collected from wild-type C57BL/6 J mice according to previously published methods^{20 (link)}. In brief, total RNA was extracted with Qiagen RNeasy Plus Micro RNA isolation kit (Qiagen, Germantown, MD, USA) following the manufacturer’s protocol. One sample equaled the tissue pooled from both eyes of each animal. cDNA was synthesized using Ready-To-Go First-Strand beads (GE Healthcare Life Sciences, Marlborough, MA, USA) and random hexamers (Life Technologies, Grand Island, New York, USA) with 1 µg total RNA as template. DNA concentration was measured with a Qubit spectrophotometer (Life Technologies). Digital polymerase chain reaction (PCR) was performed as previously described with a QuantStudio 3D Digital PCR system (Life Technologies) with Slc5a8 Taqman assay primer set Mm00520629_m1 (Applied Biosystems, Inc. [ABI], Foster City, CA) and normalized by concentration of cDNA^{33 (link)}.

Total RNA from conjunctiva or the corneal epithelium was extracted using a QIAGEN RNeasy Plus Micro RNA isolation kit (Qiagen) following the manufacturer’s protocol. Conjunctiva was surgically excised. One sample equaled the tissue pooled from both eyes of each animal. After isolation, the concentration of RNA was measured and cDNA was synthesized using the Ready-To-Go™ You-Prime First-Strand kit (GE Healthcare) as previously described15 (link). Real time PCR was performed using specific Taqman probes for IL-17A (Il17a, Mm0043918_m1), IFN-γ (Ifn-γ, Mm00801778_m1), IL-13 (Il13, Mm99999190_m1), Foxa2 (Foxa2, Mm00839704_mH), Integrin α2 (Itga2 or DX5, Mm00434371_m1) (Taqman Universal PCR Master Mix AmpErase UNG) in a commercial thermocycling system (StepOnePlus™ Real-Time PCR System, Applied Biosystems), according to the manufacturer’s recommendations. The beta-2 microglobulin (β2 m) (B2m) (Mm00437762_m1) gene was used as an endogenous reference for each reaction. The results of quantitative PCR were analyzed by the comparative C_t method in which the target of change = 2⁻ΔΔ^Ct and were normalized by the C_t value of β2 m and the mean C_t of relative mRNA level in the untreated naïve group. Gene expression of 3–4 mice per group per time point in two independent experiments with a total of 6–8 mice was determined.

For quantitative polymerase chain reaction (QPCR) studies, epithelium was scraped using a dulled blade and frozen immediately. Four corneas per sample and at least 3 samples per time point were used for the QPCR studies. RNA was extracted using a QIAGEN RNeasy Plus Micro RNA isolation kit (Qiagen) following the manufacturer's protocol. After isolation, the concentration of RNA was quantified using a NanoDrop^® ND-2000 Spectrophotometer (Thermo Scientific, Wilmington, DE) and stored at −80 °C until used. QPCR was performed using a Bio-Rad CFX384 Real-Time PCR detection system. The primers used were ordered from Bio Rad, unless otherwise specified: Bec1(qMmuCID0005981), LC3 (qMmuCED0045817), LAMP1 (qMmuCID0027030), LAMP2 (qMmuCID0011408), CXCL1(qMmuCED0047655), BDNF(qMmuCED0050333), NTN1(Qiagen #QT00128478), DCC(Qiagen #QT00135100), Unc5b(Qiagen #QT00167846), Efna4(Qiagen #QT00100681), Efna5(Qiagen #QT00116494), Rgma (Qiagen #QT00310583) and GAPDH (qMmuCED0027497). QPCR data was normalized against GAPDH.

Following euthanasia, the corneal epithelium was scraped, and total RNA was extracted using a QIAGEN RNeasy Plus Micro RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instruction. The cDNA was synthesized using the Ready-To-Go⁻You-Prime-First-Strand kit (GE Healthcare, Pittsburgh, PA, USA). Quantitative real-time PCR was performed with specific Taqman probes (Life Technologies, Grand Island, NY, USA) for STRA6 (Mm00486457_m1) and RBP-4 (Mm00803264_g1). The HPRT-1 gene was used as an endogenous reference for each reaction. The results of real-time PCR were analyzed by the comparative CT method.

For quantitative polymerase chain reaction (qPCR) studies, epithelium was scraped using a dulled blade and was frozen immediately in liquid N2. Four corneas per sample and at least three samples per time point were used for the qPCR studies. RNA was extracted using a QIAGEN RNeasy Plus Micro RNA isolation kit (Qiagen, Germantown, MD., USA), following the manufacturer’s protocol. Two mice per age were pooled and RNA was isolated. After isolation, the concentration of RNA was quantified using a NanoDrop^® ND-2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and stored at −80 °C until used. qPCR was performed using a Bio-Rad CFX384 Real-Time PCR detection system. The primers used were ordered from Bio Rad, unless otherwise specified: Bec1(qMmuCID0005981), LC3 (qMmuCED0045817), LAMP1 (qMmuCID0027030), LAMP2 (qMmuCID0011408), CXCL1 (qMmuCED0047655), BDNF (qMmuCED0050333), NTN1 (Qiagen #QT00128478), DCC (Qiagen #QT00135100), Unc5b (Qiagen #QT00167846), Efna4 (Qiagen #QT00100681), Efna5 (Qiagen #QT00116494), Rgma (Qiagen #QT00310583), and GAPDH (qMmuCED0027497). qPCR data are normalized against GAPDH.

Conjunctival epithelium was excised from B6 and Pinkie strains and total RNA was extracted using a QIAGEN RNeasy Plus Micro RNA isolation kit (Qiagen) according to the manufacturer's instructions. The concentration and purity of RNA was assessed using a NanoDrop 1,000 (ThermoFisher Scientific, Waltham, MA). RNA-Seq was performed by the Beijing Genomics Institute (BGI) using the BGISEQ500RS to generate 100-bp paired-end reads. The sequencing reads were cleaned by removing reads containing adapter or poly-N sequences, and reads of low quality using SOAPnuke (version 1.5.2, parameters: -l 15 -q 0.2 -n 0.05). and the expression levels of the resulting genes and transcripts were determined using RSEM (version 2.2.5, default parameters). A total of 19,511 genes were obtained as raw data. Detection of DEGs (differentially expressed genes) was performed with DEseq2 (Parameters: Fold Change >2.00 and adjusted P < 0.05). Genes were passed through the Benjamini-Hochberg procedure to obtain the critical value for false discovery and a total of 1,375 genes passed with a P >0.0006. The selected genes in the IL-17 signaling pathway were clustered in a heat map using GraphPad Prism 9.0 software (San Diego, CA, USA).