PCR Profiler array analysis of various gene expression related to autophagy

The Human Custom RT2 Profiler™ PCR Array (CAPH11870A, Qiagen) profiles the expression of 84 genes involved autophagy. RNA was purified using the Qiagen RNAse kit, including on-column DNAse treatment to remove genomic DNA. cDNA was prepared with the RT2 First Strand Kit (SA Biosciences, Frederick, Maryland, USA). A PCR profiler array was performed (RT2 SYBR Green/ROX qPCR Master Mix; SA Biosciences) in 96-well plates on an ABI 7300 instrument (Applied Biosystems, California, USA). For data analysis, the ΔΔCt method was applied using the RT2 Profiler PCR Array software package, and statistical analyses performed (n = 3). This package uses ΔΔCT–based fold change calculations and the Student’s t test to calculate two-tail, equal variance P-values. The fold changes were calculated using the equation 2^−ΔΔCt. If fold change was greater than 1, the result was considered as fold up-regulation. If fold change was less than 1, the negative inverse of the result was considered as fold down-regulation³⁴.