Kapa sybr fast universal 2x qpcr master mix

Total RNA from cells was extracted using RNeasy mini kit (Qiagen, Hilden, Germany), retrotranscribed and amplified using Applied Biosystems High-Capacity cDNA Reverse Transcription kit (Life Technologies) as per manufacturer’s instructions. RT-PCR was performed using KAPA SYBR FAST Universal 2X qPCR Master Mix and Qiagen QuantiTect Primer Assays using standard procedures in a LightCycler 480. Threshold cycles (Ct) were calculated using the LightCycler^® 480 software (all Roche, Basel, Switzerland). Relative quantification using the comparative Ct method was used to analyse the data output. Values were expressed as fold change over corresponding values for the control by the 2^−∆∆Ct method. Primers were from QIAGEN: GAPDH (Cat# QT00079247), GFAP (Cat# QT00081151), nestin (Cat# QT00235781), SOX2 (Cat# QT00209685), TUBB3 (Cat# QT00083713).

Total RNA from cells was extracted using RNeasy mini kit (Qiagen, Hilden, Germany), retrotranscribed and amplified using Applied Biosystems High-Capacity cDNA Reverse Transcription kit (Life Technologies) as per manufacturer’s instructions. RT-PCR was performed using KAPA SYBR FAST Universal 2X qPCR Master Mix and Qiagen QuantiTect Primer Assays using standard procedures in a LightCycler 480. Threshold cycles (Ct) were calculated using the LightCycler^® 480 software (all Roche, Basel, Switzerland). Relative quantification using the comparative Ct method was used to analyse the data output. Values were expressed as fold change over corresponding values for the control by the 2-ΔΔCt method. Primers from QIAGEN: GAPDH, CDKN1A, ORC1, MCM5, NEK2, BUB1, N2RF1. Customized primers (Integrated DNA Technologies, Coralville, IO, IA): p27 (fwd: 5′-CTG ATG CTG TTG CTC GGT TA-3′; rv: 5′-TGC AGA CTC TGG GAC ATC TG-3′), DEC (fwd: 5′-GGT TAG CGG AGC AAT GCG CA-3′; rv: 5′-AAC CGG CAT TTG GGG AAC CGT C-3′).