Human autophagy rt2 profiler pcr array

Karpas-299 cells were treated or not for 24 h with 500 nM crizotinib, then washed once with PBS and harvested. Frozen cell pellets were sent to SABiosciences (Hilden, Germany), where both the RNA extraction and the Human Autophagy RT² Profiler PCR array were performed to study the expression profile of 84 key genes involved in autophagy. Amplification data (fold changes in C_t values of all the genes) were analyzed by the ΔΔC_t method. Data were normalized to controls (PBS-treated cells), assigned as 1.

The Human Autophagy RT2 Profiler PCR Array (SABiosciences, PAHS-084Z) was used to study autophagy-specific gene expression profiles in accordance with the manufacturer's recommendations. Briefly, total RNA was isolated from different experimental groups using Trizol (Invitrogen, 15596-018). Potential genomic DNA contamination was removed from samples by treatment with RNase-free DNase (Invitrogen, 79254) for 15 min at 37°C. The RNA concentration and purity were determined using a NanoDrop ND-1000 (Thermo Scientific). First-strand cDNA was synthesized from 1–2 μg total RNA using SuperScript III Reverse Transcriptase (Invitrogen, 18080044). After cDNA synthesis, real-time PCR was performed using SuperArray PCR master mix (Qiagen, 330503) and a Roche LightCycler® 480 (96-well Block) according to the manufacturer's instructions. Amplification data (fold-changes in Ct values of all genes) were analyzed using the ΔΔCt method.

The Qiagen RT² Profiler PCR Array Human Autophagy was used to measure the expression levels of autophagy‐associated genes. This PCR array system is a reliable and accurate tool for analyzing the expression of 84 genes associated with autophagy machinery components (autophagic vacuole formation, vacuole targeting, protein transport, autophagosome‐lysosome linkage, ubiquitination, and proteases) and the regulation of autophagy (coregulators of autophagy and apoptosis, coregulators of autophagy and the cell cycle, autophagy induction by intracellular pathogens, autophagy in response to other intracellular signals, and chaperone‐mediated autophagy). Moreover, five housekeeping genes, one genomic DNA control, three reverse transcription controls, and three positive PCR controls were included in the same 96‐well plate.
The procedure began with the conversion of each 0.5 μg RNA sample into first‐strand cDNA using the RT² First Strand Kit. Next, the cDNA was mixed with an appropriate RT² SYBR^® Green Master mix. This mixture was aliquoted into the wells of the RT² Profiler PCR Array. PCR was performed, and relative expression was determined using data from the real‐time cycler and the ΔΔCT method.

The expression levels of 84 key genes in autophagy were determined by RT² Profiler™ PCR Array Human Autophagy (SABiosciences-Qiagen, Hilden, Germany). Total RNA was isolated from cultured cells, and 1 μg of total RNA was reverse-transcribed to singlestranded cDNA using the RT2 First Strand kit (SABiosciences-Qiagen, Hilden, Germany). The expression levels of genes of interest were determined by quantitative PCR using CFX96 Touch™ Real-Time PCR Detection System (Bio Rad Laboratories, Inc., Hercules, CA, United States) with SYBR Green fluorophore using the RT² SYBR Green Master Mix (SABiosciences-Qiagen, Hilden, Germany). The reaction program involved 40 cycles of 95°C for 10 min, 95°C for 15 s and 60°C for 1 min. The results were analyzed using the manufacturer’s software and relative gene expression was quantified using the 2^−ΔΔCq method (Livak and Schmittgen, 2001 (link)). The altered expression of the 84 genes was displayed using heat imaging with normalization to ACTB, B2M and GAPDH.