Abi prism 7900 sequence

Total RNA was extracted from mouse liver, primary ECs, or MCECs using the Qiazol reagent (Qiagen, Valencia, CA), and treated with DNase I (Zymo Research, Inc.) to remove trace amounts of contamination by genomic DNA. The cDNA from each RNA sample was synthesized using a high-capacity cDNA reverse transcription kit supplied by Applied Biosystems (Applied Biosystems, Inc.). The cDNA was subsequently used for real-time quantitative PCR to measure gene expression levels. We used SYBR Green I dye (SYBR Green PCR kit; Bio-Rad) for real-time quantitative PCR with the ABI Prism 7900 sequence detection system (Applied Biosystems) (16 (link), 17 (link)). The primers were designed using GenScript Real-time PCR Primer Design software, with primers crossing exon junctions. The nucleotide sequences of each primer were Blast searched against the GenBank database to confirm the uniqueness of each primer. The primers used for this study are shown in supplemental Table S1. The concentration of each primer was optimized to avoid dimer formation. The Ct value of each sample was normalized with the endogenous housekeeping β-actin gene, expressed as relative quantification (RQ). RQ is power of 2^−ΔCt.

TRIzol reagent (Invitrogen) was used to extract total RNA according to the manufacturer’s instructions. A NanoDrop 2000 (Thermo Fisher Scientific) was applied to measure the quantity and purity of the total RNA. The PrimeScript RT Master Mix kit (TAKARA) was used to synthesize cDNA. Real-time PCR was performed using FastStart Universal SYBR Green Master Mix (Rox) (Roche) in an ABI PRISM 7900 sequence detector (Applied Biosystems, Carlsbad, CA), and GAPDH was applied as an internal control. Relative mRNA levels were computed according to the Ct values relative to GAPDH. Supplementary Table 2 shows the primers used in this study.

Complementary DNA (cDNA) was generated from 500 ng RNA using Promega reagents as described [18 ] and diluted 75-fold in RNAse-free water for subsequent analysis. qRT-PCR was performed in a 13 μL reaction with 5 μL of diluted cDNA, 6.5 μL of 2x TaqMan or SYBR Green reagent (Applied Biosystems), 1.3 μL of 3 mM forward and reverse primer mix (including 1.5 mM of probe for TaqMan reactions) and 0.2 μL of RNAse-free water according to the default manufacturer's protocol (Applied Biosystems). Primer sequences are listed in Supplementary Table S1. Reactions were run in duplicate for each sample and quantified using the ABI Prism 7900 sequence detection system (Applied Biosystems). Duplicates were checked for reproducibility, and then averaged; ‘no reverse transcriptase’ controls were included to check for genomic DNA contamination, and ‘no template’ controls were included to check for the formation of primer dimers. Product specificity was determined using a dissociation curve for SYBR green reactions. A standard curve generated from a pool of all cDNA samples was used for quantification. The expression of genes of interest was normalized using the BestKeeper method to the geometric average of 3–4 housekeeping genes (18s, Actb, 36b4 and Tbp), and data were expressed as arbitrary units or normalized to the average of control group.