Quantitative RT-PCR Gene Expression Analysis

qRT-PCR using power SYBR green (Thermo Fisher Scientific) was performed as previously described (Smith et al., 2013 (link)) using an Applied Biosystems 7500 Fast Real-Time PCR System. All primers (Table 1) were tested for efficiency using serial dilutions, and results were normalized to 18S RNA levels; data analyses were performed using the standard curve analysis method (Rutledge and Côté, 2003 (link); Smith et al., 2014 (link)). The 18S ribosomal transcript was used as a housekeeping gene as our hypoxia treatments did not alter its expression (Table 2).

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Kiernan E.A., Ewald A.C., Ouellette J.N., Wang T., Agbeh A., Knutson A.O., Roopra A.S, & Watters J.J. (2020). Prior Hypoxia Exposure Enhances Murine Microglial Inflammatory Gene Expression in vitro Without Concomitant H3K4me3 Enrichment. Frontiers in Cellular Neuroscience, 14, 535549.

Publication 2020

power SYBR green 7500 Fast Real-Time PCR System

18s rna Dilutions Housekeeping gene Hypoxia Primers Ribosomal Sybr green

Corresponding Organization : University of Wisconsin–Madison

Top 5 similar protocols

Variable analysis

independent variables

Hypoxia treatments

dependent variables

Gene expression levels

control variables

18S ribosomal transcript expression (as a housekeeping gene)

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