Gene Expression Analysis of Stem Cell Markers

The total RNA was extracted using the Qiagen mRNA extraction kit, according to the manufacturer’s instructions (Qiagen, Manchester, United Kingdom). RNA quality and quantity were assessed by nanodrop analysis. The cDNA was generated by the reverse transcriptase reaction and used for real-time PCR (qRT-PCR). The following genes were studied: NSE, Asl1, Hes6, Nanog, Twist, Snail, E-cadherin, and Wnt-11 (corresponding primer sequences given previously [32 (link),33 (link)]). Analysis by real-time qPCR was performed using Taq SYBR Green premix (Qiagen, Manchester, United Kingdom), as reported before. Relative levels of mRNA expression were calculated using the Comparative CT/2-ΔΔCT method [33 (link),34 (link)]. RPII (RNA polymerase II) was used as the reference gene. [34 (link)] Experiments were performed three times as biological repeats with triplicate technical repeats, then statistical significance was analysed using a Student’s t-test.

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Arisan E.D., Rencuzogullari O., Keskin B., Grant G.H, & Uysal-Onganer P. (2020). Inhibition on JNK Mimics Silencing of Wnt-11 Mediated Cellular Response in Androgen-Independent Prostate Cancer Cells. Biology, 9(7), 142.

Publication 2020

Qiagen mRNA extraction kit Taq SYBR Green premix

Asl1 Biological Cdna E cadherin Gene Mrna Primer Real time pcr Reverse transcriptase Rna polymerase ii Snail Student Sybr green

Corresponding Organization : University of Westminster

Other organizations : Gebze Technical University, Istanbul Kültür University, University of Bedfordshire

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Variable analysis

independent variables

Extraction method: Qiagen mRNA extraction kit

dependent variables

MRNA expression levels of the following genes: NSE, Asl1, Hes6, Nanog, Twist, Snail, E-cadherin, and Wnt-11

control variables

RPII (RNA polymerase II) was used as the reference gene

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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