Total cellular RNA was isolated by the modified method of Chomczyński [63 (link)] from the obtained cells using TRI Reagent (Sigma), isopropanol (Sigma), chloroform (Sigma), ethyl alcohol (Poch, Poland). The RNA extract was spectrophotometrically evaluated.
The reverse transcription reaction was carried out as recommended by the manufacturer, using the High-Capacity reagent cDNA Transcription Kits with RNase Inhibitor (Applied Biosystems, USA) and 1 μg of isolated RNA.
Relative gene expression levels of NAIP, BIRC2, BIRC3, BIRC5, BIRC6, BIRC7, BIRC8, XIAP, XAF1, OCT4 and SOX2 were examined by the qPCR method using commercially available TaqMan probes (Applied Biosystems, USA): GAPDH: Hs99999905_m1; for NAIP gene: Hs03037952_m1; for BIRC2 gene: Hs00357350_m1; for BIRC3 test gene: Hs00154109_m1; for BIRC5: Hs00153353_m1; for BIRC6: Hs00212288_m1; for BIRC7: Hs01086675_m1; for BIRC8: Hs01057786_s1; for XIAP: Hs00236913_m1; for XAF1: Hs00213882_m1; for OCT4: Hs00242302_m1; and for SOX2 gene: Hs00193931_m1. GAPDH was an endogenous control gene. The expression of the examined genes was calculated from the formula RQ = 2−ΔΔCT [64 (link)]. The Expression Suite Software 1.0.3 was used to calculate gene expression level (Life Technologies). Detailed procedures for the RNA isolation and qPCR reaction shave have been described in our previous work [23 (link),48 (link)].
The reverse transcription reaction was carried out as recommended by the manufacturer, using the High-Capacity reagent cDNA Transcription Kits with RNase Inhibitor (Applied Biosystems, USA) and 1 μg of isolated RNA.
Relative gene expression levels of NAIP, BIRC2, BIRC3, BIRC5, BIRC6, BIRC7, BIRC8, XIAP, XAF1, OCT4 and SOX2 were examined by the qPCR method using commercially available TaqMan probes (Applied Biosystems, USA): GAPDH: Hs99999905_m1; for NAIP gene: Hs03037952_m1; for BIRC2 gene: Hs00357350_m1; for BIRC3 test gene: Hs00154109_m1; for BIRC5: Hs00153353_m1; for BIRC6: Hs00212288_m1; for BIRC7: Hs01086675_m1; for BIRC8: Hs01057786_s1; for XIAP: Hs00236913_m1; for XAF1: Hs00213882_m1; for OCT4: Hs00242302_m1; and for SOX2 gene: Hs00193931_m1. GAPDH was an endogenous control gene. The expression of the examined genes was calculated from the formula RQ = 2−ΔΔCT [64 (link)]. The Expression Suite Software 1.0.3 was used to calculate gene expression level (Life Technologies). Detailed procedures for the RNA isolation and qPCR reaction shave have been described in our previous work [23 (link),48 (link)].
Free full text: Click here
