Quantitative RT-PCR and Protein Analysis

For qRT-PCR assays, cell and tumor tissue RNA was extracted according the manual of PrimeScript RT reagent Kit (Takara Biotechnology Co., Ltd., China) and Primers were present in Additional file 1: Table S1. The StepOnePlus Real Time PCR System (Applied Biosystems, Foster City, CA, USA) was used. The resulting data were analyzed with the comparative cycle threshold (CT) value for relative gene expression quantification relative to GAPDH.
Cell proteins were extracted using normal method [7 (link)]. After blocking, antibody against HIF-1α (Abcam, UK), Nestin, SOX2, VEGF and GAPDH (Proteintech, China), were added, respectively.

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Zeng X., Wang Q., Tan X., Jia L., Li Y., Hu M., Zhang Z., Bai X., Zhu Y, & Yang X. (2019). Mild thermotherapy and hyperbaric oxygen enhance sensitivity of TMZ/PSi nanoparticles via decreasing the stemness in glioma. Journal of Nanobiotechnology, 17, 47.

Publication 2019

PrimeScript RT reagent Kit StepOnePlus Real Time PCR System antibody against HIF-1α Nestin VEGF GAPDH

Assays Blocking antibody Cell Gapdh Gene expression Nestin Primers Proteins Sox2 Tissue Tumor rna Vegf

Corresponding Organization :

Other organizations : Huazhong University of Science and Technology

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Variable analysis

independent variables

PrimeScript RT reagent Kit (Takara Biotechnology Co., Ltd., China)
Primers listed in Additional file 1: Table S1
Antibodies against HIF-1α (Abcam, UK), Nestin, SOX2, VEGF and GAPDH (Proteintech, China)

dependent variables

Relative gene expression quantification relative to GAPDH
Cell and tumor tissue RNA
Cell proteins

control variables

StepOnePlus Real Time PCR System (Applied Biosystems, Foster City, CA, USA)
Normal method [7 (link)] for cell protein extraction

positive controls

GAPDH

negative controls

Not explicitly mentioned

Annotations

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