Sybr green pcr master

Total RNA was extracted from cells with TRIzol Reagent (Invitrogen). Reverse transcription was conducted with the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland) and qRT-PCR was conducted using SYBR Green PCR Master (Roche) to measure mRNA expression. Primers for Bod1 and the GAPDH internal control were purchased from GeneCopoeia. For miRNA detection, reverse transcription and qRT-PCR was performed using an ALL-in-one™ miRNA qRT-PCR Detection Kit (GeneCopoeia). Primers for miR-142-3p and the U6 internal control were also purchased from GeneCopoeia. qRT-PCR was performed using a Bio-Rad Chromo4 System RealTime PCR detector (Bio-Rad, USA). All procedures were conducted according to the manufacturers' protocols. Relative fold-change in mRNA expression was calculated using the 2−^ΔΔCt method with the following equation: RQ (Relative Quantitation) = 2^−ΔΔCt.

qRT-PCR was performed using a cDNA Reverse Kit and an SYBR Green PCR Master (Roche, Basle, Switzerland) according to the manufacturer's instruction. Semi-quantitative analysis of relative gene expression was performed on a CFX96TM Real-Time PCR Detection System (Bio-Rad, Redmond, WA, USA). GAPDH was used as the housekeeping gene to standardize the amount of mRNA expressed. Details of the 27 mRNAs detected in this study as well as the primer sequences are listed in Table S1.

Total RNAs of 2D- and 3D-cultured cells were extracted with TRIzol Reagent (Life Technologies, USA). For mRNA detection, reverse transcription was carried out with Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland), and qRT-PCR was carried out with SYBR Green PCR Master (Roche, Switzerland). The primers of OCT4, SOX2, NANOG, c-MYC, LIN28 and internal control of GAPDH were purchased from GeneCopoeia (Guangzhou, China). For miRNA detection, reverse transcription and qRT-PCR was performed by using an ALL-in-one^TM miRNA qRT-PCR Detection Kit (GeneCopoeia, Guangzhou, China). The primers of miR-302a and internal control U6 were also purchased from GeneCopoeia. Q-PCR was performed on samples using a Bio-Rad Chromo4 System Real-Time PCR detector (Bio-Rad, USA). All steps were carried out according to the protocol. The relative fold change of mRNA was calculated by using the 2^−ΔΔCt method.

Total RNA was extracted with TRIzol reagent (Life Technologies). Afterwards, total RNA (500 ng) was reverse-transcribed using High-Capacity cDNA Reverse Transcription Kit (4368813, Thermo Fisher Scientific). QRT-PCR assay was performed with 1 μL cDNA, 2 μL primers mix, and SYBR Green PCR Master (4913914001, Roche, Switzerland) by a 7500 Fast Real-Time instrument (Applied Biosystems, USA). MRNA expression level was normalized to GAPDH gene, and miRNA level was normalized to RNU6 gene. The primer pair sequences used in the present study are listed in Supplementary Table 1.