Taqman gene expression assay primer

RNA extraction was made using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) following the manufacturer’s instruction. On-Column DNase I Digestion Set (Sigma-Aldrich) was used to eliminate genomic DNA from RNA extracts. RNA was converted to cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer’s instruction. qPCR assays were performed in the Rotor-Gene 6000 system (Corbett Research) using TaqMan™ Fast Universal PCR Master Mix (2X), no AmpErase™ UNG (Applied Biosystems), TaqMan™ Gene Expression Assay primers and probe with the following ID: Hs00356857_m1 (FUT3), Hs01106466_s1 (FUT4), Hs00704908_s1 (FUT5), Hs03026676_s1 (FUT6), Hs00237083_m1 (FUT7), Hs00276003_m1 (FUT9), Hs00327091_m1 (FUT10) and Hs00543033_m1 (FUT11). The relative mRNA levels were normalized against the mean of the β-actin (Hs99999903_m1) and GAPDH (Hs99999905_m1) expression and calculated by adapted formula 2^−ΔCt × 1000, which infers the number of mRNA molecules of the gene of interest per 1000 molecules of the endogenous controls [88 (link)]. ΔCt stands for the cycle threshold difference between the target gene and the endogenous control genes. Cycle thresholds were obtained by analyzing the qPCR results with the Rotor Gene 6000 software.

Total RNA was prepared using QIAGEN's RNeasy Micro kit (QIAGEN, 27220 Turnberry Lane Suite 200 Valencia, CA 91355) according to the manufacturer's instructions. For first‐strand cDNA synthesis, 1 μg of total RNA, 20 pmol of Oligo (dT)12–18, and 200 units of SuperScript II Reverse Transcriptase (Invitrogen) were mixed in a final volume of 20 μL. Synthesized cDNA (1 μL) was added to a 20‐μL PCR mixture containing TaqMan Gene Expression Assay primers (Applied Biosystems, 850 Lincoln Centre Drive Foster City, CA 94404, USA.) and TaqMan Universal PCR Master Mix. Each sample was amplified in triplicate. PCR consisted of 40 cycles of denaturation at 95 °C for 15 s, annealing, and amplification at 60 °C for 60 s in an ABI7900HT Sequence Detection System machine (Applied Biosystems). The specific primers for the TaqMan Gene Expression Assay primers were as follows: PUMA: Mm00519268_m1; BAX: Mm00432050_m1; p21:Mm00432448_ml; FasL: Mm00438864_ml; Caspase 3: Mm01195085_ml. p16: Mm01257348_m1 p73:Mm00660220_ml. Bcl2:Mm00477631_m1. Bcl‐xL:Mm00437783_m1. 18S rRNA primers (4319413E or Mm00519571_ml) were used as internal controls.

The validity of the microarray results was tested via quantitative real-time PCR (qRT-PCR) employing the StepOne Plus qPCR machine (Applied Biosystems, Life Technologies, Carlsbad, California, USA). For validation we selected seven genes [NEXN (Hs00332124_m1), GCC2 (Hs00206083_m1), ROCK 1 (Hs01127699_m1), ROCK2 (Hs00178154_m1), DNM1 (Hs00247147_m1), KTN1 (Hs00192160_m1), and C12ORF39 (Hs00228976_m1)] which showed a fold change (FC) > 0.5 in the microarray. GAPDH (Hs99999905_m1) and β-actin (4326315E) were selected as reference genes for normalization. cDNA of the samples was used for quantification. TaqMan Gene expression Assay primers, probes and gene expression master mix (all Applied Biosystems, Life Technologies, Carlsbad, CA, USA) were used to run the qRT-PCR according to manufacturer's instructions. Mean plate efficiencies were calculated by LinReg.

Total RNA was isolated from MCC cells via RNeasy Kit (Qiagen) per manufacturer’s instructions. Complementary DNA was generated from MCC mRNA using High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Quantitative real-time-PCR (qRT-PCR) was performed with a StepOne Plus Real-Time PCR System (Applied Biosystems) as described previously using specific TaqMan Gene Expression Assay primers purchased from Applied Biosystems: PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ and MRPS2 (mitochondrial ribosomal protein S2). Triplicate runs of each sample were normalized to MRPS2 mRNA to determine relative expression.

Our microarray gene expression results were confirmed by RT-qPCR using three biological replicates of the four independent samples applied to the microarray analyses. Commercially available TaqMan Gene Expression Assay primers for the target genes, Cux2 (Mm00500377_m1), Wfs1 (Mm00495979_m1), Mbp (Mm01266402_m1), and Rprm (Mm00469773_s1), Ctgf (Mm01192933_g1), and the reference gene, Gapdh (Mm99999915_g1), were obtained from Applied Biosystems (USA).