High capacity rna to cdna reagents

RNA was isolated from MEF monolayers using TRIzol (Invitrogen). Genomic DNA was eliminated from RNA samples by digestion with RNase-free DNase (Promega). RNA concentration was determined by NanoDrop and equivalent amounts for each sample were used for cDNA synthesis using High Capacity RNA-to-cDNA reagents (Applied Biosystems). Quantitative PCR analysis was performed to detect expression of collagen type I alpha 1 chain (Col1a1), collagen type II alpha 1 chain (Col2a1), and collagen type III alpha 1 chain (Col3a1) in a 7500 thermal cycler (Applied Biosystems) using forward and reverse primers (Col1a1 forward 5′-GCATGGCCAAGAAGACATCC-3′; Col1a1 reverse 5′-CCTCGGGTTTCCACGTCTC-3′; Col2a1 forward 5′-AGAACAGCATCGCCTACCTG-3′; Col2a1 reverse 5′-CTTGCCCCACTTACCAGTGT-3′; Col3a1 forward 5′-TCAAGTCTGGAGTGGGAGG-3′; Col3a1 reverse 5′-TCCAGGATGTCCAGAAGAACCA-3′) and Fast SYBR Green PCR Master Mix (Applied Biosystems). Each sample was run in triplicate and normalized to GAPDH followed by normalization to respective collagen content of Acvr1^+/+ on day 1.