Three hundred naograms of total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s protocol. qPCR was performed in 384-well format31 (link) on a QuantStudio 5 qPCR machine (Thermo Fisher). Expression of all genes was first normalized to the levels of the reference gene YWHAZ, and then plotted relative to the levels of YWHAZ (i.e., 1.0 = equivalent to YWHAZ). Forward and reverse primer sequences used to detect the expression of the respective genes are provided in Supplementary Table 1 .
All qPCR experiments were performed once. For each qPCR experiment, two biological replicates (two different wells in the same culture plate) were analyzed; qPCR was then performed on two technical replicates for each biological replicate (i.e., 2 biological replicates × 2 technical replicates = 4 replicates altogether). Plotted qPCR data represents the average of the 4 replicates, and standard error is shown.
All qPCR experiments were performed once. For each qPCR experiment, two biological replicates (two different wells in the same culture plate) were analyzed; qPCR was then performed on two technical replicates for each biological replicate (i.e., 2 biological replicates × 2 technical replicates = 4 replicates altogether). Plotted qPCR data represents the average of the 4 replicates, and standard error is shown.
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