Cfx96 touch real time pcr detection system 1

For confirmative RT-qPCR, 3,000-cell fractions from 2C, LZ, PS and RS populations were sorted as explained above, but using a MoFlo Astrios EQ (Beckman Coulter) in Purify mode. Generation of cell lysates, reverse transcription and qPCR were performed using Power SYBR Green Cells-to-Ct kit (Ambion, Life Technologies) following the instructions of the manufacturer. For qPCR step, 2 µL cDNA in 20 µL final volume reaction mix was used. All the reactions were made in a CFX96 Touch Real-Time PCR Detection System 1 (BioRad, Hercules, CA), with three biological replicas each.
As the expression levels of commonly used control genes such as Gapdh and Actb significantly vary across the different testicular cell populations (e.g. see Fig. 5I), we chose Surf4 (Surfeit gene 4) as normalizing gene because it exhibited similar expression levels in the four cell populations in our RNAseq data (55.99 ± 2.91 FPKM in 2C; 59.91 ± 10.31 FPKM in LZ; 59.36 ± 5.76 FPKM in PS; 45.09 ± 4.38 FPKM in RS). The coding genes and their AS lncRNAs or neighbour lincRNAs selected for confirmation by RT-qPCR are shown in Fig. 5, and all especially designed primers are listed in Supplementary Table S6.
Amplification efficiency of the primers was >93%. We made relative expression quantification using the 2^−ΔΔCt method, and 2C as calibrator condition.