For detection of special candidate genes, stemness and EMT markers quantitative PCR after reverse transcription of mRNA was used as previously described (17 (link)). Total mRNA was isolated 24 h after seeding by using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed with the help of the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). For qRT-PCR TaqMan® probes were used and the SensiFASTTM Probe Lo-ROX Kit (Bioline, Memphis, Tennessee, USA). qPCR was conducted with the Viia7 RUO thermocycler (Applied Biosystems). mRNAs of the target genes were detected with TaqMan® assays (all Thermo Fisher Scientific, Waltham, MA, USA) as described in detail in the Supplementary Material and Methods section. Values of target genes were normalized to mean values of both housekeeping genes and calculated with the comparative cycle threshold method (2-ΔCt). Finally, each data set was normalized to the value of nd cells.
Viia7 ruo thermocycler
Manufactured by Thermo Fisher Scientific
The Viia7 RUO thermocycler is a real-time PCR system designed for research use. It features a 96-well block format and supports a wide range of sample volumes from 10 to 100 microliters. The system is capable of performing quantitative, endpoint, and melt curve analysis.
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2 protocols using viia7 ruo thermocycler
1
Quantitative Gene Expression Profiling
2
RT-qPCR analysis of gene expression
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For RT-qPCR total mRNA was isolated from HeLa cells using the RNeasy® Mini Kit, including DNase treatment, following reverse-transcription with QuantiTect® Reverse Transcription Kit (all from Qiagen).
Total genomic DNA was employed for qPCR. The primers used (from Thermo Fisher and Biomers) are listed inSupplementary Table 1 . Both RT-qPCR and qPCR were performed using KAPA SYBR® FAST qPCR Kit (peqlab/VWR) on a Viia7 RUO thermocycler (Applied Biosystems Life Technologies). Data was analysed by Viia7 RUO software version 1.2.1. mRNA expression of target genes was calculated with the 2−ΔΔCt method, whereby TATA box binding protein (TBP) transcript levels served as an internal control. For 7S and mtDNA analysis by qPCR the 2ΔCt method was used and 18S rRNA served as internal control.
Total genomic DNA was employed for qPCR. The primers used (from Thermo Fisher and Biomers) are listed in
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