Norgen all in one kit

Cell populations were subjected to qPCR analysis of OTX2, LIN8A, NANOG, SOX2, OCT4, MMP1, TIMP4, CNTN1, UNC5C, FOS, FOSB and IGF1 transcript levels. Total RNA was extracted using the Norgen all-in-one kit (Norgen Biotek) according to the manufacturer's guidelines. First-strand cDNA was synthesized using the Superscript III First Strand Synthesis System (Invitrogen). The following PCR conditions were used: 50°C for 2 min, 95°C for 2 min, and 40 cycles of 95°C for 15 s, 60°C for 30 s. Quantitative PCR was conducted using GoTaq qPCR Master Mix (Fisher Scientific) and performed on a Mx3000P (Stratagene) qPCR system. All values were normalized to GAPDH. Specific primer sequences for each gene are listed in supplementary material Table S6.

Daoy, UI226 and/or MED311 tumorspheres were dissociated, and counted using an automatic cell counter and stained for CD271. Sort samples were also stained with 7AAD viability dye (Beckman Coulter). Cells were sorted on the basis of CD271+ and CD271− expression using a MoFlo XDP cell sorter. Analysis of cell sorting was performed using FlowJo software. Total RNA was extracted from Daoy control, CD271 OE, sorted Daoy CD271+/−, sorted MED311 CD271+/− cells, as well as MED311 and UI226 KD cells using the Norgen All in one kit (Norgen Biotek, Thorold, ON, Canada) according to manufacturer's guidelines. First-strand cDNA was synthesized using the Superscript III First Strand Synthesis System (Life Technologies)). The following PCR conditions were used: 50°C for 2 minutes, 95°C for 2 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. qPCR was conducted using GoTaq qPCR Master Mix (Fisher Scientific) and performed on an Mx3000P (Stratagene, Santa Clara, CA, USA) qPCR system. All values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Specific primer sequences for each gene are listed in Table S1.

Whole RNA was extracted from Group 3 MB cells using a Norgen-all-in-one kit (Norgen Biotek, Thorold, ON, Canada). The Superscript III First Strand Synthesis kit (Life Technologies) was used for first-strand cDNA synthesis. qPCR was performed using the GoTaq qPCR Master Mix (Fisher Scientific, Ottawa, ON, Canada) and data were analyzed on a Mx3000 P Stratagene qPCR system, using the following parameters: 50 °C for 2 min, 95 °C for 2 min, and 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Primer sequences for each specific gene evaluated are listed in Supplementary Table 2 and values were normalized to GAPDH.