Abi stepone real time system

Real-time polymerase chain reaction (RT-PCR) was used to measure the expression levels of S100B using GAPDH as internal control. Total RNA was isolated using TRIzol reagent (Invitrogen, USA). The obtained RNA samples were reverse transcribed according to instructions. Primer sequences used were S100B forward: 5′-GAGCAGGAAGTGGTGGACAAA-3′, S100B reverse: 5′-CACTCCCCATCCCCATCTT-3′, GAPDH forward: 5′-GTATGACTCTACCCACGGCAAGT-3′, and GAPDH reverse: 5′-TTCCCGTTGATGACCAGCTT-3′. The amplification of S100B and GAPDH fragments was performed using the SYBR Green PCR master mix. A 10 μL PCR reaction volume was containing 5 μL SYBR green reaction mix (Invitrogen, USA), 0.5 μL sense and antisense primers, and 1 μL cDNA. The PCR reaction conditions were 95°C for 10 mins followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All reactions were performed using the ABI-StepOne Real-Time System (Applied Biosystems, USA). Dissociation curve analysis confirmed the amplification of primer specific products. Relative change in gene expression was determined using the 2^−ΔΔCt method [23 (link)].

After transfection for 24 h, cells were collected, and total RNA preparations were extracted using TRIzol reagent (Invitrogen). We used a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States ) for assessment of RNA concentration and quality. Extracted RNA was reverse transcribed into complementary DNA with the P5X All-In-One MasterMix Kit (Abmgood, Vancouver, BC, Canada) according to the manufacturer’s instructions. Levels of mRNAs of FOXA2 were measured in samples using SYBR premix ex Taq Kit (Takara, Tokyo, Japan) in an ABI Step One Real-Time system (Applied Biosystems, Waltham, MA, United States ). The relative expression of mRNA to that for β-actin was calculated using the 2^−∆∆Ct method. The primers used for RT–qPCR are listed in Supplementary Table S4.

RNAs were purified from various tissues of mice using a NucleoSpin RNA kit (Macherey–Nagel) and then reverse-transcribed using SuperScript III (Invitrogen). Comparative ΔC_T method was applied to identify relative changes in mRNA expression using SYBR Premix (Applied biosystems, 4,367,659) and ABI StepOne real-time system (Applied biosystems) with the corresponding primer sets: mBmp10 (#1: 5′-ACA TCA TCC GGA GCT TCA AGA ACG-3′, 5′-AAC CGC AGT TCA GCC ATG ACG-3′; #2: 5′-GCA GAT GAG GTC GAA CAT GA-3′, 5′-GGC CTG AAT AAT TGC GTG TT-3′; #3: 5′-GGA TCC ACC AGA GTA CAT GCT-3′, 5′-GTC CAC GCC ATC ATA CAT CA-3′), Actin (5′-CCT GAA CCC TAA GGC CAA CCG-3′, 5′-GCT CAT AGC TCT TCT CCA GGG-3′). The expression levels were normalized to Actin as indicated.