Taqman mutation detection assay

Total RNA was extracted from hybrid ESCs and NPCs using the miRNEasy Kit (Qiagen) and DNaseI treatment (Qiagen). cDNA was synthesized from 5 μg of RNA using random hexamers and SuperScript III (ThermoFisher) reverse transcriptase. Meg3 and Dlk1 expression were quantified by RT-qPCR using SYBR Green I master mix (Roche) on a Lightcycler 480 instrument. Mean C_T values were normalized with the mean of two housekeeping genes (Actb, Gapdh) and the ΔΔCt method [54 (link)]. Primer sequences are in Additional file 1: Table S2.
The Taqman mutation detection assay was used for allele-specific quantitation (ThermoFisher). Expression was quantified using Taqman Genotyping Master Mix (ThermoFisher) and a Lightcycler 480 instrument. The levels were normalized to two housekeeping genes (Actb, Gapdh) amplified with SYBR Green Master Mix (Roche), as reported before [27 (link)].

To assess the presence of EGFR mutation (EGFR driver and T790M mutation) in plasma, approximately 20 mL of whole blood was collected using K2-EDTA collection tubes; within 4 h after blood collection, the samples were then centrifuged to separate the plasma from the peripheral blood cells at 1500 ×g for 10 min at 4 °C, and the plasma supernatant was transferred to conical tubes and stored at − 80 °C until transport. The plasma samples were transported at − 80 °C to one of two commercial laboratories (SRL Inc., Tokyo, Japan, and G&G Science Inc., Fukushima, Japan). The cobas EGFR Mutation Test v2 (Roche Molecular Systems, CA, USA) was used to detect EGFR mutations in the extracted plasma cfDNA. The mutant allele frequency and EGFR copy number were measured using the QX200 Droplet Digital PCR System (Bio-Rad, Hercules, CA). Isolated cfDNA was amplified using ddPCR Supermix for Probes (Bio-Rad) using EGFR T790M/L858R (PrimePCR ddPCR Mutation Assay; Bio-Rad), EGFR del19 assay (TaqMan Mutation Detection Assay; Thermo Fisher Scientific) for del19 EGFR mutations, and EGFR (RPP30 reference gene) for gene CNV (PrimePCR ddPCR Copy Number Assay; Bio-Rad), according to the manufacturers’ protocols. The ddPCR data were analyzed using Quanta Soft analytical software (version 1.7.4, Bio-Rad).

DNA was isolated from cell lines by Purelink genomic DNA mini kit (Thermo Fisher Scientific). EGFR tyrosine kinase domains (exon 18–21) in PC9 and PC9/ER cells were amplified by PCR and mutations were detected with Sanger sequencing. For detection of T790M in each cell lines, TaqMan Mutation Detection Assay (Thermo Fisher Scientific) was also performed.
EGFR-exon 18
Forward 5′-CAAATGAGCTGGCAAGTGCCGTGTC-3′
Reverse 5′-GAGTTTCCCAAACACTCAGTGAAAC-3′
EGFR-exon 19
Forward 5′-GCAATATCAGCC TTAGG TGCGGCTC-3′
Reverse 5′-CATAGAAAGTGAACATTTAGGATGTG-3′
EGFR-exon 20
Forward 5′-CCATGAGTACGTATTTTGAAACTC-3′
Reverse 5′-CATATCC CCATGGC AAACTCTTGC-3′
EGFR-exon 21
Forward 5′-CTAACGTTCGCCAG CCATAAGTCC-3′
Reverse 5′-GCTGCGAGCTCACCCAGAATGTCTGG-3′

Competitive Allele-Specific TaqMan PCR (castPCR) was performed to detect and measure somatic mutation of BRAFV600E using the TaqMan Mutation Detection Assay (Hs00000111_mu, Applied Biosystems) for c.1799T>A in BRAF (RefSeq accession no: NM_004333.4). A “B-Raf V600E Genomic DNA Reference Standard” sample (Horizon Diagnostics, https://www.horizondiscovery.com) was used as positive control in a 1:2 dilution series of six samples (corresponding to an allelic frequency range: 0.78–50%), and a blood donor gDNA was used as a negative control. Mutation detection by castPCR was carried out in 10 μL reactions in 384-well format, each well comprising 20ng gDNA template, 1X Genotyping Master Mix, and 1X TaqMan Mutation Detection Assay (containing allele-specific forward primer, locus-specific TaqMan probe, locus-specific reverse primer, allele-specific MGB blocker), and run on an Applied Biosystems QuantStudio Real-Time PCR System using the following thermal cycling conditions: 95°C for 10 minutes; 5 cycles: 92°C for 15 seconds and 58°C for 1 minute; 40 cycles: 92°C for 15 seconds and 60°C for 1 minutes. All samples were run in triplicates. The mutation status and allele frequency (AF) was analyzed using Mutation Detector TM software (Applied Biosystems). Each sample was considered as positive (AF>0.1%) or negative (AF = 0%) for the BRAFV600E mutation.

Genomic DNA was isolated from frozen skin tissues by DNAzol (MRC, Inc., Charleston, WV, USA) according to the protocol provided by the manufacturer. CastPCR was performed with TaqMan Mutation Detection Assay designed to detect CAA → CTA transversion in codon 61 of the mouse H-ras gene (Applied Biosystems by Life Technologies, Foster City, CA, USA) following the manufacturer’s instruction. The castPCR was run on a GeneAmp 7300 Sequence Detection system (Applied Biosystems, Foster City, CA, USA) using the universal mutation detection thermal-cycling protocol. The mutational status of a sample was determined by calculating the ΔCt value between the mutant allele assay and wild-type allele assay to obtain the percent mutation according to manufacturer’s instruction.

For detection of mutant allele IDH1 c.395G>A (p.R132H, COSMIC ID 28746), TaqMan Mutation Detection Assays (assay name: IDH1 28746mu and IDH1 rf) with the TaqMan Mutation Detection IPC Reagent Kit (Life Technologies, Carlsbad, California, USA) were used. Mutant allele detection was performed according to recommended procedure and reaction conditions in the manual. For the amplification, the Stratagene Mx3000P realtime PCR system instrument (Agilent Technologies, Inc., Santa Clara, CA, USA) was used. Detection of mutant alleles was performed in duplicate in a reaction volume of 20 μL. Detection of reference gene was also performed in duplicate. The analysis of positive samples was repeated. Before analysis of collection of tumor samples, the samples of normal brain tissue were analyzed for detection of cut-off amplification curve. No amplification of mutant allele was present in normal brain tissue. On the basis of these results and the shape of amplification curve of positive tumor samples, the 25 ΔCt cut-off value was determined.