Sybr green qpcr pre mix

The β-glucan fraction (BGF) was obtained from the carpophores of P. baumii as described previously [16 (link)]. Briefly, dried and powdered carpophores were extracted with distilled water at 100 °C for 10 h. The filtered extract was concentrated in vacuo, and three volumes of ethanol were added at 4 °C for 4 h to precipitate the crude BGF. The crude BGF was purified by passing through a Sephacryl S-400 HR column (Sigma-Aldrich, St. Louis, MO, USA) and BGF was obtained as a strong single band. The β-glucan content in the BGF was 84% by analysis with a mushroom and yeast assay kit (Megazyme, Bray, Ireland). β-Glucans of yeast zymosan A from Saccharomyces cerevisiae and barley were obtained from Sigma-Aldrich. Mouse diets containing 1% normal and 10% high-salt were obtained from Samtako (Osan, Korea).
The polymerase chain reaction (PCR) primers for corin and GAPDH were synthesized by Cosmogenetech (Seoul, Korea). The forward and reverse primer sequences (5′→3′) of corin were 5′GTC CGC ATT ATT CCT CTG GA3′ and 5′ CAA ACC AGA GGA CCA CCA CT3′, respectively. Those of GAPDH were 5′ ACC GCA GCT AGG AAT AAT GGA ATA 3′ and 5′ CTT TCG CTC TGG TCC GTC TT 3′, respectively. Oligo deoxythymine, 5X reaction buffer, Moloney murine leukemia virus (M-MLV) reverse transcriptase, and SYBR green qPCR PreMIX were obtained from Enzynomics (Daejeon, Korea).

For qPCR analysis, total RNA was solubilized in TRIzol reagent (Invitrogen) and extracted according to the manufacturer’s instructions. cDNA was synthesized from 1 mg of total RNA using reverse transcription, and the amount of mRNA was determined using real-time PCR analysis with the SYBR Green qPCR Pre Mix (Enzynomics) on an ABI real-time PCR 7500 machine (Applied Biosystems). Samples were normalized to TBP or 18S ribosomal RNA. The primer sequences are described in Supplementary Table S1.