Rt2 rna qpcr kit

GSE-standardized preparation was a gift from Kikkoman Corp. (Nado City, Japan). The preparation composition is as follows: 89.3% procyanidins, 6.6% monomeric flavanols, 2.24% moisture content, 1.06% of protein, and 0.8% of ash, as reported recently (Derry et al., 2013 (link), Velmurugan et al, 2010a (link), 2010b (link)). Purchased antibodies include anti-CDX2, anti-CK20, anti-Surfactant D and anti-TTF-1 (all from Abcam). Anti-mouse and anti-rabbit horseradish peroxidase (HRP) secondary antibodies were from Invitrogen (Carlsblad, CA) and Cell Signaling Technology (Beverly, MA). RNA was isolated via the Qiagen RNeasy Kit, amplified via the Qiagen RT² RNA qPCR kit. Additionally, RNA transcript was quantified via specific RNA TaqMan primers for Cdx2 from Life technologies (Grand Island, NY) and primers for mouse Ck20 from Invitrogen.

Total RNA was isolated (from 20mg tissue in each case) employing Qiagen RNeasy Kit, as per vendor’ protocol, and RNA concentration was determined with a NanoDrop 2000 (Thermo Scientific). Next, Qiagen RT² RNA qPCR kit was used following manufactures protocol and the First Strand cDNA Synthesis Reaction was stored at −20°C. In qRT-PCR, the commercially available and pre-validated TaqMan primer/probe set used was: Cdx2 (CDX2; Mm01212280_m1). The primer set for Ck20 was designed 5′-GCGTTTATGGGGGTGCTGGAG-3′ (F) and 5′-AAGGCTTGGGCGGTGCGTCTC-3′ (R). mRNA levels of triplicate samples from each group were measured by real-time quantitative reverse transcription-PCR using ABI PRISM 7700 at the Molecular Biology Shared Resources of the University of Colorado Cancer Center. Quantities of specific mRNA in each sample were normalized to the corresponding 18S rRNA levels to determine overall transcriptional modification.