Taqman pre developed assay reagent

The Applied Biosystems™ TaqMan™ Pre-Developed Assay Reagents from Thermo Fisher were used for real-time relative quantitative evaluation of mouse Stxbp1 gene expression. Experiments were performed according to manufacturer’s protocol. Briefly, the anti-mouse Stxbp1 probe (Mm00435837_m1) and anti-mouse GAPDH probe (Glycerinaldehyd-3-phosphat-Dehydrogenase; 4352932E) purchased from Applied Biosystems™ (by Life Technology) were diluted 1:20 and used to detect murine Stxbp1 gene segments. GAPDH expression served as reference. All samples were run in duplicates using a Quantstudio-7 machine from applied Biosystems (Thermo Fisher).

Quantitative RT-PCR was performed as previously described [26 (link)]. TaqMan gene expression assays (Life Technologies) were used for UCK1 (Hs01075618_m1) and UCK2 (Hs00367072_m1). The TaqMan Pre-Developed Assay Reagent (Life Technologies) was used for ACTB. The relative expression level of each gene to the ACTB expression level was determined by the ΔCT method.

Quantitative RT-PCR was performed as previously described (Ohyashiki et al., 2005 (link)). Total RNA was purified from AZA treated cells, shHP1γ transfected cells or negative control cells incubated in the presence of 1 μg/ml doxycycline for 96 h. TaqMan gene expression assays (Life Technologies Inc., Carlsbad, CA, United States) were used for CBX5 coding HP1α (Hs01127577_m1), CBX1 coding HP1β (Hs01080635_g1), and CBX3 coding HP1γ (Hs04234989_g1). TaqMan Pre-Developed Assay Reagent (Life Technologies Inc., Carlsbad, CA, United States) was used for ACTB. The expression level of CBX1,3 and 5 relative to the ACTB expression level was determined by the ΔCT method.

Collected cells from all 3 cell lines were pelleted and total RNA isolation was performed using the RNeasy Plus Mini Kit (Cat # 74136, Qiagen, USA). Quantification of mRNA was done by spectrophotometry (260/280 nm ratio, NanoDrop 1000, Thermo Scientific). The high capacity RNA-to-cDNA kit (Cat # 4387406, Applied Biosystems, USA) was used for reverse transcription. Primer/probe sets were obtained as Taqman pre-developed assay reagents (concentrated and pre-optimized mix of primers and FAM-labeled Taqman probe) from Applied Biosystems (University Park, IL). SYBR green gene primer sets with gene specific forward and reverse primers were used for the human cell lines. The specific primers that were designed for gene amplification are listed and described in Additional file 1: Table S1. Reaction volumes were 25 μL, and 40 reaction cycles were run for each gene in an Applied Biosystems 7300 Real-Time PCR System. We used the comparative cycle threshold (ΔΔC_T) method to determine relative gene expression, with each gene normalized to either β-ACTIN (human cells) or hypoxanthine guanine phosphoribosyltransferase (Hprt1) (murine cells) expression. Results are reported as fold change over control [(2^−ΔΔCT)]. A fold-change > ± 1.5 with a p-value < 0.05 was considered significant.

RNA from cell lines and tissue samples were extracted using RNeasy (Qiagen, CA, USA), and 100 ng of RNA was converted to cDNA using TaqMan Reverse Transcription Reagents Kit (Applied Biosystems, CA, USA). qPCR was performed using 0.5 ng of cDNA and TaqMan Pre-Developed Assay Reagents (Applied Biosystems, CA, USA) under recommended conditions on a Mx3005P instrument (Agilent Technologies, Santa Clara, USA). All samples were run in triplicates, and the relative expression was calculated using the equation RQ = 2^−ΔΔCT. CT values > 35 were regarded as negative. RPS29 has been shown to be stably expressed in adult human testis and germ cell neoplasms^{40 (link)} and was used as a reference gene in our study. The primers used are listed in Supplementary Table S2.

The probes for Alb (GenBank accession no. NM_009654.3; Mm00802090_m1), Afp (GenBank accession no. NM_007423.4; Mm00431715_m1), Tat (GenBank accession
no. NM_146214.3; Mm01244282_m1), Tdo2 (GenBank accession no. NM_019911.2; Mm00451269_m1), Cyp1a2 (GenBank accession no. NM_009993.3; Mm00487224_m1), Cyp2e1 (GenBank accession no. NM_021282.2; Mm00491127_m1), Cyp2a5 (GenBank accession no. NM_007812.4; Mm00487248_g1), Cyp7a1 (GenBank accession no. NM_007824.2; Mm00484150_m1), Lgr5 (GenBank accession no. NM_010195.2; Mm00438890_m1), Otc (GenBank accession no. NM_008769.3; Mm00493267_m1) and Pck1 (GenBank accession no. NM_011044.2; Mm01247058_m1) genes were obtained from TaqMan Pre‐Developed Assay Reagents (Applied Biosystems). A mouse Gapdh (GenBank accession no. NM_008084.2) TaqMan probe (4352339E, Applied Biosystems) was included as an endogenous control. The RT products (2 μl) were then subjected to real‐time PCR analysis in a final volume of 10 μl, with the use of TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and with an ABI 7500 Thermocycler. Each sample was assayed in triplicate.