Applied biosystems 7300 fast real time pcr system

TRIzol reagent (Invitrogen, CA, USA) was used to isolate total RNA from the cells following the manufacturer’s guidelines. The RNA was converted into cDNA by reverse transcription using the microRNA First-Strand cDNA Synthesis Kit (Sangon Biotech, Shanghai, China) and RevertAid First Strand cDNA Synthesis Kit (Sangon Biotech, Shanghai, China). Real-time PCR was performed to detect the RNA expression using SYBR Green PCR Master Mix (Applied Biosystems) and running with the Applied Biosystems 7300 Fast Real-Time PCR System (Applied Biosystems). The miRNA expression level was standardized to U6, and the mRNA expression level was standardized to GAPDH. The results were analyzed by the 2^−△△Ct method. The primer sequences used in this study are described in Table.1.Table 1

Primer sequences used in this study

Primer name	Primer sequence
GAPDH	F: 5′-GGTTGTCTCCTGCGACTTCA-3′
	R: 5′-TGGTCCAGGGTTTCTTACTCC-3′
LICAM	F: 5′-TGCTCCTCATCCTGCTCATCCTC-3′
	R: 5′-TCACTGTACTCGCCGAAGGTCTC-3′
mmu-miR-214-3p	F: 5′-TACAGCAGGCACAGACAGGC-3′

The qPCR reactions were performed using an Applied Biosystems 7300 Fast Real-Time PCR System (Applied Biosystems, USA) with primers for total yeast, Saccharomyces, Hanseniaspora, and Starmerella bacillaris as described in Andorrà et al. (2010 (link)). Standard curves were built for each yeast species in triplicate using 10-fold serial dilutions of fresh cultures.

Total RNA sample (1000 ng) was reversely transcribed to cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Darmstadt, Germany). Amplification and qPCR process were performed using SYBR Green PCR kit (TaKaRa, Dalian, China) on Applied Biosystems 7300 Fast real-time PCR system (Applied Biosystems, Waltham, MA, USA). β-actin was used as endogenous control. The sequences of primers used in PCR are listed in Table S1.

Expression levels of specific genes (Table 1) were determined using qPCR. Total RNA from 1 × 10⁷ cells was isolated using a PureLink^® RNA Mini kit from Ambion Life Technologies (Woburn, MA, United States) as recommended by the manufacturer. To remove DNA, a DNAse (Qiagen, Barcelona, Spain) step was performed at 37°C for 15 min before washing. Reverse transcription and qPCR reactions were performed as described by Beltran et al. (2004) (link). cDNA was synthesized from 320 ng/μL RNA using SuperScript^® III Reverse Transcriptase (Invitrogen) and Oligo (dT) 20 Primer (Invitrogen). qPCR was performed using the Applied Biosystems 7300 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, United States). Samples were prepared as follows: 2 μL cDNA, 0.4 μL each primer, 0.4 μL ROX, 10 μL SYBR Green (Takara^® SYBR Green master mix), and H₂O q.s.p. 20 μL. Relative gene expression was calculated using the 2^-ΔΔCt formula, where Ct is defined as the cycle at which fluorescence is determined to be statically significantly above background; ΔCt is the difference in Ct of the gene of interest and the housekeeping gene (ACT1), and ΔΔCt is the difference between ΔCt of the condition MEL at 6 h or 16 h and the Control at 6 h or 16 h (see figure legends for relative expression details). Three biological replicates were analyzed for each time point and condition.

Total RNA was extracted from 16HBE cells with the RNAiso Reagent (TaKaRa, Japan). First-strand cDNA was synthesized from 2 μg total cellular RNA with the PrimeScript RT reagent Kit (TaKaRa). To amplify PCDH1, specific primers were designed based on the gene sequences. Gene-specific primer sets were designed for human PCDH1 isoforms 1 and 2 as follows: PCDH1 isoform 1, 5′-GACTCTTCCAGATTGGGTCACAT-3′ and 5′-CTTGCCGCGGTCACTGA-3′; PCDH1 isoform 2, 5′-TGCCAATGCAGAAATCGAATAC-3′ and 5′-CGGGCCCTGAACAGTGAT-3′. Primers for amplification of GAPDH were used as an internal control: 5′-CAAGTTCAACGGCACAGTCAAG-3′ and 5′-ACATACTCAGCACCAGCATCAC-3′. The Applied Biosystems 7300 Fast Real-Time PCR System with SYBR green PCR master mix (Applied Biosystems) were used according to manufacturer protocols. The reactions were incubated in a 96-well optical plate at 95 °C for 20 s, followed by 40 cycles each of 95 °C for 3 s and 60 °C for 30 s. The threshold cycle (Ct) data were obtained using default threshold settings. Ct is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.