Abi prism 7500

The procedure undertaken has been described in Chopy et al. (2011). Basically, cDNA synthesis was performed with 1 μg total RNA using SuperScript II reverse transcriptase (Life Technologies, France). Quantitative real‐time RT‐PCR (qRT‐PCR) was performed in triplicate using an ABI Prism 7500 fast sequence detector system (primers 18S: F: CTT AGA GGG ACA AGT GGC G, R: ACG CTG AGC CAG TCA GTG TA; α7 AchR QT00074732, Qiagen, France) with GoTaq PCR master mix (Promega, Charbonniéres‐les‐Bains, France). After normalization to 18S rRNA, the relative abundance of mRNA was obtained by calculation of the difference in threshold cycles of the test and control samples (mock value set to 1), commonly known as the ΔΔC_T method.

Real-Time was performed on cDNA obtained accordingly to High Capacity c-DNA Reverse Transcription Kit (Applied BioSystem, CA, USA). PCR reactions were performed with 75–300 ng of cDNA, using an ABI Prism 7500 and Quantitect SYBR Green PCR kit & Quantitexct Primer Assay (Qiagen Gmbh, Hilden, Germany), as published [63 (link)].

Total RNA was isolated from fresh villous tissue and HTR-8/SVneo cells using the RNeasy Mini Kit (Qiagen, USA). RNA was quantified using spectrophotometric absorbance readings and treated with DNase I (Qiagen) to exclude DNA contamination. Then 1 µg of RNA was reverse transcribed with the Reverse Transcription Kit (Qiagen). An ABI PRISM 7500 real-time PCR system and miScript SYBR Green PCR Kit (Qiagen) were used to perform polymerase chain reactions. The amplification program consisted of 1 cycle of initial incubation at 50 °C for 2 minutes and 95 °C for 10 minutes, followed by 50 cycles of 95 °C for 15 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds, and a final extension at 72 °C for 5 minutes. The primers used in the study are listed in Table S1. GAPDH was used as an internal control for mRNA. U6 was used as an internal control for miRNA. Relative RNA expression was calculated by the standard 2^-ΔΔCtmethod.