Total RNA was purified with an RNA preparation kit (RNeasy Plus Mini Kit; Qiagen) and a homogenizer (QIAshredder; Qiagen). Paired-end RNA deep sequencing analysis was performed by the University of Michigan DNA genomic core following quality assessment using the TapeStation system (Agilent). qPCR and duplex qPCRs were performed as previously described (34 (link)) using gene-specific primers and fluorescent dye–labeled probes (Applied Biosystems Life Technologies). Reactions were performed and monitored using a real-time PCR system (CFX384; Bio-Rad). Relative normalized mRNA levels were calculated using the ΔΔCt method.
Fluorescent dye labeled probes
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluorescent dye–labeled probes are laboratory reagents used to detect and quantify specific target molecules or sequences in various biological samples. They typically consist of a short, single-stranded nucleic acid sequence labeled with a fluorescent dye that emits light upon excitation, allowing for the visualization and analysis of the target of interest.
Automatically generated - may contain errors
2 protocols using fluorescent dye labeled probes
1
RNA-Seq and qPCR Analysis Protocol
2
Quantitative RT-PCR Analysis of Retinal Gene Expression
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Retinas and cell pellets were collected, flash-frozen in liquid nitrogen, and stored at −80°C until analysis. Total RNA was purified with an RNA preparation kit (RNeasy Plus Mini Kit; Qiagen, Venlo, Limburg, The Netherlands), and a homogenizer (QIAshredder; Qiagen) for dissociation of retinal tissues. Quantitative real-time (qRT)-PCR and duplex qPCRs were performed as previously described.13 (link) Duplex qPCRs were performed using gene-specific primers and fluorescent dye-labeled probes (Applied Biosystems Life Technologies, Carlsbad, CA, USA). Primer-probe assay information and gene information are provided in Supplementary Table S1 . Reactions were performed and monitored using a real-time PCR system (CFX384; Bio-Rad, Hercules, CA, USA). Relative normalized mRNA levels were calculated using the ΔΔCt method.
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