Applied biosystem 7500 fast real time pcr system

A total of five significant genes differentially methylated in recurrent versus non-recurrent samples CCNE1 (EPSH107315-1A), DDX43 (EPSH112573-1A), CHL1 (EPSH110000-1A), PON3 (EPSH113282-1A), and CCNDBP1 (EPSH104489-1A) were selected from the DNA methylation profile for validation using EpiTect Methyl II PCR assays (SABiosciences) following the manufacturer’s protocol. Methylation-sensitive (EPHS115450-1A) and methylation-dependent (EPHS115451-1A) digest control assays were performed on 34 fresh tissue samples (31 non-recurrent and 3 recurrent tumors). Digested DNA was used as template for qPCR assay using RT² SYBR Green ROX qPCR Mastermixes (SABiosciences, #330520) under standard amplification conditions on Applied Biosystem 7500 Fast Real-Time PCR system (Applied Biosystems, Inc.). Data generated by MS-qPCR were further analyzed as recommended by the manufacturer¹. Meanwhile, two FFPE samples of recurrent tumor were validated using methylated and unmethylated primers designed from AIT Biotech, Singapore. The bisulfite conversion was performed using EpiTect Bisulfite Kit (SABiosciences) according to the manufacturer’s instruction. MS-qPCR was carried out using Methylamp ^TM MS-qPCR Kit (Epigentek Group, Inc.) according to the manufacturer’s protocol on Applied Biosystem 7500 Fast Real-Time PCR system (Applied Biosystems, Inc.).

Total RNA was extracted from the erlotinib-sensitive and -resistant cell lines using the total RNA purification plus kit (Norgen Biotek Corporation, Thorold, ON, Canada) and retro-transcribed with the GoScript Reverse Transcription System (Promega Italia, Srl, Milan, Italy) using random primers. Quantitative real time PCR (qRT-PCR) analysis was performed in an Applied Biosystem 7500 Fast Real-Time PCR System (from Thermo Fisher Scientific, Milan, Italy) using Applied Biosystem PowerUp SYBR Green Master Mix for qPCR. Sequences of specific primers are shown in Table S11. Ribosomal protein L31 (RPL31) was used as a reference gene to normalize the quantitation of target genes for differences in the amount of total RNA in each sample. The relative fold change of target genes in resistant cell lines in comparison to HCC827 and HCC4006 parental cell lines was calculated using the 2^−ΔΔCt method. The data were analyzed using Applied Biosystem SDS (Ver. 1.4) software (Thermo Fisher Scientific, Milan, Italy).

RNA samples, prepared from either whole cells or gradient fractions by TRIsure™ reagent (Meridian Bioscience, Cincinnati, OH, USA), were reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Carlsbad, CA, USA), according to the manufacturer’s guidelines. For RNA19 transcript analysis, primers were as follows: 16S.FOR, 5′-TATACCCACACCCACCCAAG-3′; and ND1.REV, 5′-GCGATTAGAATGGGTACAAT-3′. Cterm RNA assessment was carried out with the following primers: Cterm.FOR, 5′-AAATTCCTGTGCCCCAACAA-3′; and FLAG.REV, 5′-CTACTTATCGTCGTCATCCT-3′. The reference transcript was amplified with hs18S.FOR, 5′-GTAACCCGTTGAACCCCATT-3′; and hs18S.REV, 5′-TGAAGAACAAATTCGTGGACTTTG-3′.
Amplification reactions were carried out using the SsoAdvanced Universal SYBR^® Green Supermix (BioRad, Hercules, CA, USA) and analysed with the Applied Biosystem 7500 Fast Real-Time PCR System (ThermoFisher Scientific, Carlsbad, CA, USA). Relative quantification of the qPCR products was achieved using the Pfaffl method [22 (link)]; statistical analysis was performed using the unpaired two-tailed Student’s t test.