Quantstudio 6 flex real time pcr system software

Next, 100,000 MCF10A cells were seeded in six-well plates in full medium. Treatments were added and repeated every 3–4 days. After 9 days, cultured cells were washed, trypsinized, and collected. Total RNA was extracted with a NucleoSpin RNA II kit (Macherey Nagel), following the manufacturer’s protocol specifications. Total RNA quantity and quality were determined using a NanoDrop spectrophotometer (N1000, Thermo Fisher Scientific, Waltham, MA, USA). RNA reverse transcription to double-stranded cDNA was performed using SuperScript™ VILO™ cDNA Synthesis Kit (Invitrogen™, Waltham, MA, USA), according to the manufacturer’s protocol, followed by incubation in the thermocycler for the reaction. Real Time (rt)-PCR was performed with a QuantStudio 6 Flex instrument (Applied Biosystems, Waltham, MA, USA) by using Fast SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA), and analysis was conducted on QuantStudio 6 Flex Real-Time PCR System software. Oligonucleotide sequences of analyzed genes, namely KRT5, KRT14, TP63, KRT8, MUC1, and HPRT1 (Sigma-Aldrich, Saint Louis, MO, USA), are listed in Supplementary Table S1. Relative quantification was performed using HPRT1 gene as a loading control. DDCT was calculated and data of each gene were analyzed using a 2^−DDCT method and reported as mean fold change.

Total RNA was extracted from HUVECs, THP-1 macrophages or peripheral blood CD14⁺ monocyte-derived macrophages, exposed to CM from FACS-sorted PCa pTA-NKs or HC pNK cells, using the small RNA miRNeasy Mini Kit (Thermo Fisher) and quantified by Nanodrop Spectrophotometer. Following genomic DNA removal, by DNase I Amplification Grade (Thermo Fisher) treatment, reverse transcription was performed on 500 ng of total RNA using SuperScript VILO cDNA synthesis kit (Thermo Fisher). Real-time PCR was performed using SYBR Green Master Mix (Thermo Fisher) on QuantStudio 6 Flex Real-Time PCR System Software (Applied Biosystems, Thermo Fisher Scientific, USA). All reactions were performed in triplicate. The GAPDH gene was used as housekeeping and results were showed as 2^^−ΔCt. HUVECs or THP-1 macrophages in their respective basal medium alone, were used as baseline controls. Primer sequences are provided in Supplementary Table 3.

Total RNA was extracted from HCM exposed to A009 (dilution 1:800) alone or in combination with 5-FU 100 μM for 24 h. The TRIzol method was used, following separation with chloroform precipitation of RNA with isopropanol (Sigma-Aldrich). The RNA pellet was washed twice with 75% ethanol (Sigma-Aldrich) and resuspended in nuclease-free water. RNA concentration was determined using the Nanodrop Spectrophotometer ND-1000 (Thermo Fisher Scientific). Reverse transcription was performed using the SuperScript VILO cDNA synthesis kit (Thermo Fisher Scientific), starting from 1,000 ng of total RNA. Quantitative real-time PCR was performed using SYBR GreenMasterMix (Applied Biosystems) on the QuantStudio 6 Flex RealTime PCR System Software (Applied Biosystems). All reactions were performed in duplicate. The relative gene expression was indicated as relative to nontreated cells, normalized to the housekeeping gene 18S. IL-6 (Fw-AGACAGCCACTCACCTCTTCAG, Rv-TTCTGCCAGTGCCTCTTTGCTG), p16 (Fw-CTCGTGCTGATGCTACTGAGGA, Rv-GGTCGGCGCAGTTGGGCTCC), and the housekeeping 18S (Fw-CGCAGCTAGGAATAATGGAATAGG, Rv CATGGCCTCAGTTCCGAAA) primers, for qPCR, were designed using the NCBI Primer BLAST tool and purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA).

The samples were treated with the TransZol Up Plus RNA Kit (ER501; TransGen, Beijing, China) for total RNA extraction, and the total bacterial RNA was subjected to reverse transcription using FastKing gDNA Dispelling RT SuperMix (KR118; Tiangen, Beijing, China).
The RT-qPCRs were carried out in a final volume of 20 μL according to the manufacturer’s instructions, using 1 μL of the cDNA dilution as template, and were mixed with 0.4 μL of forward and reverse primers. The mixture was then exposed to RT–qPCR analysis using TransStart^® Green qPCR SuperMix (AQ101-02; TransGen, Beijing, China) in the reaction in QuantStudio™ 6 Flex Real-Time PCR System Software (Applied Biosystems). Gene expression levels were measured using the 16S rRNA gene as a control using the 16SF and 16SR primers (Table 2). Gene expression levels of mutant strains were determined using the 2^−ΔΔCT method with the relative fold-difference expression against the wild-type strain. The RT–qPCR from three biological replicates was analyzed, and three technical replicates were performed.

Intact siliques were frozen in liquid nitrogen and total RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma). Total RNAs were digested with Turbo DNA-free DNase I (Ambion) according to the manufacturer’s instructions. RNA was reverse transcribed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s protocol. PCR reactions were performed in an optical 384-well plate in the QuantStudio 6 Flex System (Applied Biosystems), using FastStart Universal SYBR Green Master (Rox) (Roche), in a final volume of 10 μl, according to the manufacturer’s instructions. The following standard thermal profile was used for all PCR reactions: 95°C for 10 min, 40 cycles of 95°C for 10 s, and 60°C for 30 s. Data were analysed using the QuantStudio 6 Flex Real-Time PCR System Software (Applied Biosystems). As a reference, primers for the EIF4A cDNA were used. PCR efficiency (E) was estimated from the data obtained from standard curve amplification using the equation E = 10^−1/slope. Expression levels are presented as E^-ΔCt, where ΔCt = Ct_GOI-Ct_EIF4A. Primers are listed in S2 Table.

Total RNA was extracted using small RNA miRNeasy Mini Kit (Qiagen, Hilden, Germany). A total of 1 µg of total RNA was reverse-transcribed to cDNA using SuperScript VILO cDNA synthesis kit (Life Technologies). qRT-PCR were performed using SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Each sample was analyzed in triplicate using QuantStudio 6 Flex Real-Time PCR System Software (Applied Biosystems). The relative gene expression for pluripotency markers was expressed relative to a certified Episomal iPSC lineage (EpiPSC, Thermo Fisher Scientific), and normalized to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (Table S1).
The expression of the lentiviral vector was assessed by qualitative RT-PCR according to standard procedure. Amplified products were separated by electrophoresis on a 1% agarose gel. Primers were designed to identify cMyc (one of the four human transcription factors included in the polycistronic lentiviral backbone—forward oligonucleotide) and WPRE (woodchuck hepatitis virus post-transcriptional regulatory element, a lentiviral-specific transgene—reverse oligonucleotide) (Table S1).