Rotor gene 6000 thermal cycler

Total RNA from cells, overexpressing ORF6, was extracted by using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), and 1 $\mathsf{\mu }$ g total RNA from each sample was reverse-transcribed using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific^™, Waltham, MA, USA) according to the kit instructions. For isolation and purification of the nuclear RNA, the Cytoplasmic and Nuclear RNA Purification Kit (Norgen biotek corp., Thorold, ON, Canada) was used in accordance with the manufacturer’s instructions.
Relative expression levels of the target GFP gene was assessed by qRT-PCR analysis using the SYBR^™ Select Master Mix (Thermo Scientific^™, Waltham, MA, USA). The $\beta$ -actin housekeeping gene was used as an internal control for the normalisation of gene expression.
The sequences of the primer oligonucleotide of the studied genes are listed in Supplementary Table S1. The analysis was performed on Rotor-Gene 6000 thermal cycler (Corbett, QIAGEN, Hilden, Germany). The gene expression data were analysed by using Rotor-Gene 6000 Software (QIAGEN, version 1.8) The relative expression levels of the target genes were normalised to the endogenous control specific to each sample. Each qRT-PCR reaction was carried out with a minimum of three replicates across distinct PCR runs. Statistical significance was determined using a t-test, and significance was denoted by values less than 0.05.

Total RNA was extracted from tissues using the RNeasy Mini Kit (Qiagen) according to manufacturer's directions. Total RNA was extracted from whole blood using the QIAamp Viral RNA Mini Kit (Qiagen) according to manufacturer's directions. ZIKV-specific RNA was detected using the primers and probe set described by Faye and colleagues (Faye et al., 2013 (link)). All RT-qPCR reactions were performed on the Rotor-Gene 6000 thermal cycler (Qiagen) using the QuantiFast Probe RT-PCR Kit (Qiagen) according to manufactuer's instructions.

Total RNA was extracted using TRIzol reagent (Ambion, Warrington, UK). Cells in 12‐well plates were lysed in 600ml TRIzol reagent and transferred into Phase Lock Gel Heavy tubes (5 prime, VWR, Leicestershire, UK) according to the manufacturer's instructions. RNA purity and quantity were assessed by Nano Drop 1000 Mass spectrometer (Fisher Scientific). Ratios of A260/A280 between 1.8 and 2 were considered to be of high purity and used for cDNA synthesis. Possible contaminating DNA was removed and cDNA prepared from 1 μg RNA using QuantiTect Reverse Transcription Kit (Qiagen, West Sussex, UK) according to the manufacturer's instructions. qRT‐PCR was performed with a Rotor‐Gene 6000 thermal cycler (Qiagen) using Brilliant III Ultra‐Fast SYBR Green qPCR Master mix (Stratagene, Agilent Technologies, Cheshire, UK) and primer pairs as listed in the Table 1. PCR conditions consisted of 1 cycle of 95°C for 3 min. and 40 cycles of 95°C for 10 s. and 60°C for 10 s. followed by melting analysis of 1 cycle with gradual increase from 65 to 95°C. RPL13a was used as the housekeeping gene.

ALCAM expression was assessed by RT-qPCR. The cDNA of 44 paired LSCC and NSM was synthesized with SuperScriptIITM Reverse Transcriptase (Invitrogen^®) and quantitative PCR was carried out with the Quantifast SYBR Green PCR kit (Qiagen) in a Rotor-Gene 6000 thermal cycler (Qiagen). Gene expression quantification was performed as previously described [53 (link)]. Specific oligonucleotides were used in the expression levels analyses, as follows: ALCAM Forward 5′-AAGTGTGCAGTACGACGATGT-3′; ALCAM Reverse 5′-GGTTGCTTGAACACCTTGACT-3′; GAPDH Forward 5′ CAACAGCCTCAAGATCATCAGCAA 3′, GAPDH Reverse 5′ AGTGATGGCATGGACTGTGGTCAT 3′. RT-qPCR analyses were conducted in triplicate, using TE-1 cells as positive control, whereas negative control reactions were performed without cDNA.

Total RNA of transfected cell were extracted using Trizol reagent (Sigma, USA) and cDNA synthesis was performed using 1 µg of total RNA in 40 µl volume by random hexamer primers and MMLV reverse transcriptase kit (Takara, Japan). 50 ng of cDNA was employed for RT-qPCR to detect EGFP expression as a reporter and β-actin as a reference gene by CYBR green I (Takara, Japan) using appropriate primers (Supplementary Table 1). All reactions were performed in triplicate in the Thermal Cycler Rotor-Gene 6000 (Corbett, Australia). Vector derived EGFP expression level was measured by the comparative C_t method^{38 (link)}.

Total RNA from fibroblast cells was extracted by RNeasy Mini Kit (QIAGEN 74106) according to manufacturer’s instruction. cDNA synthesis was performed with 1 μg of total RNA implementing random hexamer primer and RevertAid™H First Strand cDNA Synthesis Kit (Takara RR037A). Real time PCR was performed with SYBR Green PCR Master Mix (TaKaRa) in a Thermal Cycler Rotor-Gene 6000 (Corbett, Mortlake, Australia), according to the manufacturer’s protocol. Expressions of target genes were normalized to B-actin gene expression level. All measurements were carried out in triplicate, from three separate samples, and data were analyzed using the 2^−ddCt method. The sequence of primers is listed in Table 3.Table 3

List of primers used in this study for real time PCR.

Gene	Forward primer (5′-3′)	Reverse primer (5′-3′)	AT*	Product size	Accession number
B-ACTIN	TTCCTGGGTATGGATCCTG	GGTGATCTCCTTCTGCATCC	58 °C	130 bp	XM_015467124.1
MTHFR	AAGATGAAGCGGAAGATG	AACCTGGAGATGAGATTG	48 °C	98 bp	NM_001011685.1
BHMT	CAGACCTTCACCTTCTATGC	CTCCTTCATCAGCCACTTG	56 °C	130 bp	NM_001011679.1
POU5F1	GGAAAGGTGTTCAGCCA	ATTCTCGTTGTTGTCAGC	62 °C	123 bp	NM_174580.3
NANOG	TTGTGACGGCTATTGTATG	ACCTCTTACTGGACTCATT	53 °C	159 bp	NM_001025344.1
DNMT3a	TGGTCCTGGGCGTTAG	CCTGCTTTATGGAGTTCG	57 °C	252 bp	NM_001206502.1
H19	TCAGCCCCGAGACCAC	ACGCTCAGAGACCAGG	53 °C	327 bp	XM_001256398.4
IGF2	CCTGCTGGAGACTTACTG	CTTGGCGAGCGTGCGA	62 °C	199 bp	XM_015461332.1