Rt2 profiler pcr arrays kit

Differentially expressed genes identified in the cDNA microarray assay were validated by quantitative RT-PCR assay by using a customized detection system, SyBr Green platform PCR Array® (Qiagen), with 88 target genes, 5 housekeeping genes, and 3 internal control genes. Validation was performed for 24 samples, three of each group (3TT, 3BT, 3BB, 3BL, 3LL, 3R1, 3R2, and 3 controls) including both samples used in the microarray assay and new samples.
The RNA samples were treated with DNase (RNase-Free DNase Set kit, Qiagen) to eliminate residual genomic DNA. Subsequently, RNA was purified and concentrated with the RNeasy MinElute Cleanup Kit (Qiagen) according the manufacturer's specification. The RT² First Strand Kit (Qiagen) was used for cDNA synthesis. The efficiency of the reverse transcription reaction was assessed by using a RT-PCR reaction for GAPDH.
The ABI ViiA 7 instrument was used for real-time PCR reactions (Applied Biosystems) following the RT2 Profiler PCR Arrays Kit (Qiagen) protocol in combination with RT2 SYBR Green Master mix (Qiagen).
The mRNAs were selected after expression analysis in all comparisons. The authors chose the most up and down regulated present in all groups, and therefore, those who were related to the disease. Others were selected because they were differentially expressed in particular forms of the disease or reaction.

After treatment, A549 cells were washed in tissue culture dish by adding cold PBS and rocking gently. Cells were washed directly in a culture dish by adding 1 ml of Trizol Reagent (Life technologies, cat. 10296–010) per 10 cm diameter dish and scraping with cell scraper. RNA was isolated according to the manufacturer's protocol. RNA concentration and purity was assessed using the nanophotomer NanodroP (Euroclone). RNA (400 ng) was subjected to reverse transcription reaction using the RT² first strand kit (Qiagen, cat.330401) according to the manufacturer's protocol.
To assess the expression of apoptotic genes, real-time quantitative reverse transcription PCR (qRT-PCR) was performed using the RT² Profiler PCR Arrays kit (Qiagen, cat.330231). Experiments were performed in triplicates for A549 cell line treated with PUAs at 5 µM concentration for 2 hours exposure time.
Plates were run on a ViiA 7 (Applied Biosystems 384 well blocks), Standard Fast PCR Cycling protocol with 10 µl reaction volumes. Cycling conditions used were – 1 cycle initiation at 95.0°C for 10 min and followed by amplification for 40 cycles at 95.0°C for 15 s and 60.0°C for 1 min. Amplification data were collected via ViiA 7 RUO Software (Applied Biosystems). The Ct-values were analyzed with PCR array data analysis online software (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php, Qiagen).