Absolutely rna nanoprep

Quantitative RT-PCR analysis was performed with gene-specific primers (Table1). Specifically, total RNA was extracted from retinas or primary cells (RGCs, astrocytes, and microglia) with Absolutely RNA Nanoprep or Microprep kits (Agilent Technologies, Santa Clara, CA, USA), and reverse transcribed with the Reverse Transcription System (Promega, Madison, WI, USA) to synthesize cDNA. To quantify the absolute number of transcripts of a gene of interest in cDNA samples, we used a standard curve. We prepared six standards, in which a gene of interest was present at 2 × 10⁵ copies, 2 × 10⁴ copies, 2 × 10³ copies, 2 × 10² copies, 20 copies, and two copies. We used a combination of 5 μL of tested cDNA samples or standards with the SYBR GREEN PCR MasterMix (Qiagen, Valencia, CA, USA) and gene-specific primers to perform qRT-PCR in the Rotor-Gene Q Cycler (Qiagen). Quantitative RT-PCR growth curves for standards and tested cDNA samples for the same gene were used to prepare a standard curve and calculate the absolute number of gene transcripts with the Rotor-Gene Q Cycler software. The absolute number of the studied gene transcripts obtained was divided by the absolute number of housekeeping gene [β-actin (Actb) or succinate dehydrogenase subunit A (Sdha)] transcripts in the same cDNA sample. These normalized data were used for further analysis.