Cfx96 real time pcr detection

Total RNA was extracted from cultured cells using TRIzol^® reagent (Invitrogen, NY). Single-stranded cDNA was generated from total RNA, using M-MLV reverse transcriptase and oligo (dT) 12–18 primers (Takara, Japan). The real-time quantitative PCR reaction was performed with the SYBR green detection system (Bio SYBR Green Master Mix, Takara, Japan). GAPDH served as an endogenous control. All cycle threshold (ct) values were determined in real time using CFX96™ Real-Time PCR Detection (Bio-rad, CA). The primer pairs for each target gene is listed in Supplementary Table 2.

Total RNA was extracted from cells using RNA purification kits (Macherey-Nagel). High capacity cDNA reverse transcription kits (Applied Biosystems) were used to generate cDNA. CFX96 real-time PCR detection (Bio-Rad) using Taqman gene expression primers TET2 (Hs0032599_m1) and GAPDH (Hs02786624g_m1) was also used. All experiments were duplicated. Gene expression was normalized to GAPDH and compared to controls. For TET1 and TET3 mRNA detection, qRT-PCR was performed by using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Cat. 1725270). Primers are listed in Supplementary Data 4.

The qRT-PCR analyses were performed using the SuperScript VILO cDNA Synthesis Kit (Life Technologies) with iTaq Universal SYBR Green Supermix (Bio-Rad). 500 ng total RNA was used in a 20 ul reverse transcription reaction. The cDNA obtained was diluted to a total volume of 100 ul and stored at -20°C. The primers for human DNMT1, DNMT3A, DNMT3B[108 (link)], DNMT2[109 (link)], XIST[110 (link)] and candidate human housekeeping gene 18S rRNA [111 (link)] were used for amplification of the target genes in normal endometrium and endometrial cancer tissues. All primers were synthesized by Integrated DNA Technologies. The qRT-PCR was performed in a 20 ul reaction consisting of 2 ul diluted cDNA, 0.2 uM of each primer and 10 ul iTaq Universal SYBR Green Supermix. All amplifications were carried out in a Bio-Rad CFX96 Real-Time PCR Detection (Bio-Rad) with denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. A melting curve analysis was performed for each run to confirm the specificity of amplification and lack of primer dimers. The qRT-PCR experiments were always run in triplicate. The relative mRNA expression levels of target genes were quantified using the 2-ΔΔCT equation for endometrial normal and cancer tissues. Mean CT of normal endometrium was used as the calibrator sample.

The qRT-PCR analyses were performed using the SuperScript VILO cDNA Synthesis Kit (Life Technologies) with iTaq Universal SYBR Green Supermix (Bio-Rad). All mouse and human brain and liver RNA was purchased from ZYAGEN. 500 ng total RNA was used in a 20ul reverse transcription reaction. The cDNA obtained was diluted to a total volume of 100ul and stored at −20 °C. The primers for human CHRNA4 and mouse Chrna4 (listed in Additional file 8: Table S3) were synthesized by Integrated DNA Technologies. The qRT-PCR was performed in a 20ul reaction mixture consisting of 2ul diluted cDNA, 0.2uM of each primer, and 10ul iTaq Universal SYBR Green Supermix. All amplifications were carried out in a Bio-Rad CFX96 Real-Time PCR Detection (Bio-Rad) with denaturation at 95 °C for 30s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30s. A melting curve analysis was performed for each run to confirm the specificity of amplification and lack of primer dimers. The qRT-PCR experiments were always run in triplicate. The relative mRNA expression levels of target genes were quantified using the 2-ΔΔCT methods as reported [45 (link)].