High capacity cdna reverse transcription reagents kit

Microarray findings were confirmed by Q-RT-PCR on select genes using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Life Technologies, Carlsbad, CA). One microgram of RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Reagents kit (Applied Biosystems) according to the manufacturer's instructions. TaqMan Universal Fast PCR master mix reagents and the Assay-On-Demand gene expression primer pair and probes (Applied Biosystems, Life Technologies, Carlsbad, CA) were used to determine the quantity of each gene in each sample. Actin beta was used as the endogenous control. Paired sample t-test were used to compare changes between Q-RT-PCR and microarray and Pearson correlation coefficient was used to examine the association between fold-change between methods.

RNA was obtained from primary trophoblasts using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s instructions. Purified RNA was treated for 1 h at 37 °C with DNas e I (DNA-free, Ambion, Austin, TX) to remove contaminating DNA. Reverse transcription to generate cDNA was performed using the High capacity cDNA reverse transcription reagents kit (Applied Biosystems) in a 50-μl reaction mix that contained 1 μg of total RNA, 1X RT buffer, 0.5 mM of each dNTP, 2.5 μM random hexamers, 0.4 units/μl RNAase inhibitor, and 1.25 units/μl Multi-Scribe reverse transcriptase, at 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 10 min. For the PCR reaction, two microliters of cDNA was used with a 300 nM each of the forward and reverse gene-specific primers (Table 2) and SYBR green PCR Master Mix (Applied Biosystems) in a total reaction volume of 20 μl. Dissociation curves were evaluated for all reactions to ensure amplification of a single product with the appropriate melting temperature. Samples were normalized to parallel reactions, which contained primers specific for YWHAZ [22 (link),23 (link)]. The fold changes of the examined genes were determined using the 2^−ΔΔCt method [24 (link)].