Quantitative real-time PCR analysis
This protocol is extracted from research article:
RBM5-AS1 promotes radioresistance in medulloblastoma through stabilization of SIRT6 protein
Acta Neuropathol Commun, Jul 5, 2021; DOI: 10.1186/s40478-021-01218-2

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Grand Island, NY, USA) following the manufacturer's instructions. Reverse transcription was performed using the SuperScript III Reverse Transcriptase kit (Invitrogen). Quantitative PCR was then done using the following PCR primers: RBM5-AS1 forward: 5′-GCTTCAACACTGCGTGACAA-3′, reverse: 5′-CGTGGAATCAAATGGAGTGG-3′; SIRT6 forward: 5′-CGTGGATGAGGTGATGTG-3′, reverse: 5′-GGCTTATAGGAACCATTGAGA-3′; CD44 forward: 5′-GCCCAATGCCTTTGATGGACC-3′, reverse: 5′-GCAGGGATTCTGTCTGTGCTG-3′; SOX2 forward: 5′-GCCTGGGCGCCGAGTGGA-3′, reverse: 5′-GGGCGAGCCGTTCATGTAGGTCTG-3′; B-Actin forward: 5′-GGTGGCTTTTAGGATGGCAAG-3′, reverse: 5′-ACTGGAACGGTGAAGGTGACAG-3′. We also performed quantitative real-time PCR arrays to profile 84 CSC-related lncRNAs in radioresistant and parental DAOY cells. The candidate lncRNAs are listed in Additional file 1: Table S1. The relative gene expression was calculated by the 2−ΔΔCt method [44].

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