Superscript first standard synthesis system for rt qpcr

Total RNA was extracted using a TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction and subsequently treated with RNase-free DNase I (Fermentas, San Diego, CA, USA). For detection of mRNA expressions, cDNA synthesis was performed from 1 µg total RNA using a SuperScript First-Standard Synthesis system for RT-qPCR (Invitrogen Life Technologies). Real-time PCR was performed by an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using Quanti-Tect SYBR Green PCR mixture (Qiagen, Hilden, Germany). The qPCR primers were: MCT1: forward: 5′-GTGGCTCAGCTCCGTATTGT-3, reverse: 5′-GAGCCGACCTAAAAGTGGTG-3′; LDHA: forward: 5′-TGGAGTGGAATGAATGTTGC-3′, reverse: 5′-ATAGCCCAGGATGTGTAGCC-3′; and β-actin: forward: 5′-AGGCACCAGGGCGTGAT-3′, reverse: 5′-GCCCACATAGGAATCCTTCTGAC-3′. For detection of miRNAs, reverse transcription reaction was performed using the TaqMan Advanced miRNA cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the instructions. qPCR was performed by ABI PRISM 7900 Sequence Detection System. The reactions were incubated in a 96-well optical plate at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. RNU6 was used as internal control. Relative expressions were calculated by the 2^−△△CT method. All reactions were performed in triplicate.

Total RNA was extracted using a TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. For detection of mRNA expressions, cDNA synthesis was performed from 1 µg total RNA using a SuperScript First-Standard Synthesis system for RT-qPCR (Invitrogen Life Technologies). Real-time PCR was performed by an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, U.S.A.). The primers were: Indian hedgehog (Ihh): forward: 5′-CAAGAAGCCCGGGATCTACA-3, reverse: 5′-GCTCGGGACTTTGTTGCTTG-3′; parathyroid hormone related protein (PTHrP): forward 5′-ACGCCCCATACAACAAAATC-3′, reverse 5′-GGTCACTGCTTGTCCAGATG-3′; and β-actin: forward 5′-GGCTGTGCTATCCCTGTACG-3′, reverse 5′-AGGTAGTCAGTCAGGTCCCG-3′. The reactions were incubated in a 96-well optical plate at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Relative expressions were calculated by the 2^−△△CT method.