Taqman 2x universal pcr master mix

To validate the miRNA sequencing results with real-time PCR, we evaluated the expression of two selected miRNAs: miR-200c-3p (ID 002300) and miR-375-3p (ID 000564). Since miR-16 emerged as equally expressed across samples in NGS data, hsa-miR-16 (ID 000391) was used as endogenous reference. In brief, 2 μL of total RNA extracted from EVs was converted into cDNA using the TaqMan microRNA RT kit (Thermo Fisher, Waltham, Massachusetts, USA), following the manufacturer’s instructions. The real-time PCR reactions were run in triplicate on an ABI 7500 real-time PCR System (Thermo Fisher, Waltham, Massachusetts, USA) using TaqMan 2X Universal PCR Master Mix (Thermo Fisher, Waltham, Massachusetts, USA), following the thermal protocol suggested by the manufacturer. Relative expression of each target was calculated by normalizing the results to the endogenous control miR-16 with the 2^−ΔCt method. The Mann Whitney U test was used to compare the expression levels of miR-200c-3p and miR-375-3p between responder and non-responder PC patients. Graphical representations were elaborated using GraphPad Prism 8 (Insight Partners, New York City, New York, USA).

Allelic discrimination assays were carried out using 25 μL total reaction, with 12.5 μL of TaqMan 2x Universal PCR Mastermix (ThermoFisher Scientific, Waltham, MA, USA), 1.25 μL primer/probe mix, and 11.25 μL of extracted DNA; the conditions were as follows: 30 s at 60 °C, 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, and 1 min at 60 °C, with a final extension step after the last cycle, for 30 s at 60 °C.
For IL-6 (rs 1800795), IL-10 (rs1800896), ACE (rs1799752,) NOS3 (rs1799980), TNF-α (rs1800629), CYP1A1 (rs2606345), and FCGR2A (rs1801274), polymorphism primers, and probes for allelic discrimination assays were ordered directly from ThermoFisher Scientific. For FUT2 (rs601338), primers and probes were designed in-house, using Primer3Plus, with probes containing zip nucleic acid (ZNA) modifications.
F: GAGGAATACCGCCACATCC, R: GGTCGTGCAGGGTGAACT, P1: HEX- CTGCTCCTGGACCTTCT-ZNA, P2: FAM -CCTGCTCCTAGACCTTCT-.ZNA.

RNA extraction was done using the RNeasy kit (QIAGEN; Cat. #74134) following manufacturer’s instructions. cDNA was synthesized from 2 μg of RNA using the High Capacity RNA-to-cDNA kit (Thermo Fisher Scientific; Cat. #4387406) following manufacturer’s instructions with a C1000 Thermal Cycler (Bio-Rad). qRT-PCR was performed with 2 μL cDNA and Taqman 2X Universal PCR Master Mix (Thermo Fisher Scientific) using a ViiA7 Real-Time PCR System (Applied Biosystems). Taqman primers were obtained from Life Technologies: Gls (Mm01257297_m1), Gls2 (Mm01164862_m1), GLS (Hs00248163_m1), and GLS2 (Hs00998733_m1). Gene expression was quantified relative to the housekeeping genes Rn18s (Mm03928990_g1) or RNA18S (Hs03928985_G1).

QRT-PCR was performed as described previously^{14 (link)}. Briefly, 10 ng of total RNA were reverse transcribed (RT) using Taqman MicroRNA RT kits and miRNA sequence-specific primers (Thermo Fisher Scientific). QPCR was carried out using Taqman 2X universal PCR Master Mix (no AmpErase UNG) along with Taqman 20X MicroRNA Assays (Thermo Fisher Scientific). Each well contained 1.33 µl of RT reaction product, 1X Taqman PCR Master Mix, and 1X specific miRNA primer, designed to detect mature miRNAs. Amplification was carried out on the ViiA^TM 7 Real Time PCR System (Thermo Fisher Scientific). Each sample was run in triplicate. Signals were normalized to small nucleolar RNA U75 (SNORDU75). U75 housekeeping miRNA run in parallel. A comparative threshold cycle (Ct) method (ΔΔCt) was used to calculate relative miRNA expression. High-throughput QRT-PCR on a custom gene panel was performed on a Fluidigm BioMark HD system in the Genomic Core at Cedars-Sinai Medical Center per manufacture instruction. Primers for miR-10b predicted target and putative LESC and corneal epithelial marker genes are listed in Supplementary Table S6. In situ hybridization was performed as described previously12 (link).

QPCR was performed as described previously [25] (link). Briefly, 10 ng of total RNA were reverse transcribed (RT) using Taqman MicroRNA RT kits and miRNA sequence-specific primers (Thermo Fisher Scientific). QPCR was carried out in MicroAmp Optical 384-well plates using Taqman 2X universal PCR Master Mix (no AmpErase UNG) along with Taqman 20X MicroRNA Assays (Thermo Fisher Scientific). Each well contained 1.33 µl of RT reaction product, 1X Taqman PCR Master Mix, and 1X specific miRNA primer, designed to detect mature miRNAs. Amplification was carried out on the ViiA 7 Real Time PCR System (Thermo Fisher Scientific). Each sample was run in triplicate. Signals were normalized to the U75 housekeeping miRNA run in parallel. A comparative threshold cycle (Ct) method (ΔΔCt) was used to calculate relative miRNA expression.