Eurogold trifast

Total RNA was extracted using EuroGOLDTriFast (EuroClone, Milan, Italy) according to the manufacturer's protocol. Reverse transcription of total mRNA was performed as described [30 (link)] starting from equal amounts of total RNA/sample (500 ng) using SuperScript® III Reverse Transcriptase (Invitrogen, Milan, Italy). Quantitative analyses of RYK, NANOG, OCT3/4, SOX2, EZH2, nestin, CD133, β-catenin, and β-actin (as an internal reference) were performed by real-time PCR using specific primers (IDT, Bologna, Italy) and iQ^TM SYBR Green Supermix (Bio-Rad). All reactions were run in triplicate. To amplificate genes of interest we used the following primers:
β-ACTIN fw:5′- TGCGTGACATTAAGGAGAAG -3′, rv:5′-GCTCGTAGCTCTTCTCCA-3′;
NESTIN fw:5′-CAGGAGAAACAGGGCCTACA -3′, rv: 5′-AGCTGAGGGAAGTCTTGGAG-3′;
EZH2 fw:5′-GAGTTGGTGAATGCCCTTGG-3′, rv:5′-TGCTGTGCCCTTATCTGGAA-3′;
OCT3/4 fw:5′-CGAAAGAGAAAGCGAACCAG -3′, rv:5′-GCCGGTTACAGAACCACACT-3′;
SOX-2 fw:5′-GCACATGAACGGCTGGAGCAAC G-3′, rv:5′-GCTGCGAGTAGGACATGCTGTAGG-3′;
NANOG fw:5′-CAAAGGCAAACAACCCACTT -3′, rv:5′-TCTGGAACCAGGTCTTCACC-3′;
RYK fw:5′-TGTAAGCTGCGAGGTCTTCA-3′, rv:5′-TTGCTGAGAAATTGCCTGTG-3′;
β-CATENIN fw:5′-TCCCACTAATGTCCAGCGTT -3′; rv:5′-ATGGACCATAACTGCAGCCT-3′;
CD133 fw:5′-TTCTTGACCGACTGAGACCC-3′, rv:5′-CCAAGCACAGAGGGTXATTG-3′.

RNA was extracted from Tet-21/N cells using EuroGold Trifast (EuroClone). cDNA was generated using Quantitec Reverse Transcription Kit (Qiagen), according to manufacturer's protocol. Quantitative analysis was performed using SYBR Green 2X PCR Master Mix (Applied Biosystem). Each sample was run in triplicate and normalized to the expression of housekeeping beta-glucoronidase (GUS) gene as previously described (34). Primers presented in Supplementary Table 1

Five million cells were lysed in EuroGold TriFast (Euroclone) and total RNA was extracted according to manufacturer’s instructions. After DNase I treatment (Life Technologies), RNA was retrotranscribed using TaqMan Reverse Transcription reagents (Roche). cDNA obtained by retrotranscription was used to confirm the presence of mutations in each cell line. To this aim, standard PCR was conducted using FastStart High Fidelity PCR System (Roche), according to the manufacturer’s instructions.
EML4-ALK fragments were amplified using forward 5′-CACAACCTCTCCAAATACACA-3′ and reverse 5′-CAGAGATCTCTGTTCGAGTC-3′ primers.
NPM-ALK fragments were amplified using forward 5′-TGCATATTAGTGGACAGCAC-3′ and reverse 5′-CTGTAAACCAGGAGCCGTAC-3′ primers.
The purity of PCR fragments was checked by agarose gel electrophoresis. Single bands were purified on column using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Otherwise the fragment was isolated from gel using the QIAquick Gel Extraction Kit (Qiagen). Purified PCR products were sequenced by standard methods at Eurofins MWG Operon.

Total RNA from mouse cerebellum was prepared using EuroGOLD Trifast (Euroclone, Milano, Italy), according to the manufacturer's instructions. One microgram of total RNA was reverse-transcribed using M-MLV (Invitrogen, Carlsbad, CA, USA) and oligo-dT primers according to the manufacturer's instructions. Total cDNA (1 μl) was used to perform qPCR using the specific primers listed in Supplementary Table 1. qPCR was performed using the iQ SYBR Green Supermix (Bio-Rad) and a C1000 Thermal Cycler CFX96 Real Time System (Bio-Rad). Expression of the gene of interest was normalized to the Gapdh house-keeping gene. Data were analyzed using the ΔΔCt method.

The mRNA expressions of MCP-1, IL-1β, TNF-α, NF-ϰB, NOX-I and NOX-II in each of the three groups of animals were analyzed with RT-qPCR and compared. RNA was extracted from brain using EuroGold Trifast (EuroClone). Quantitec Reverse Transcription Kit (Qiagen), according to manufacturer’s protocol. Quantitative analysis was performed using SYBR Green 2X PCR Master Mix (Applied Biosystem). Each sample was run in triplicate and normalized to the expression of housekeeping B-actin gene as previously described [12 (link)].

RNA was extracted from NB cells using EuroGold Trifast (EuroClone). cDNA was generated using Quantitec Reverse Transcription Kit (Qiagen), according to manufacturer's protocol. Quantitative analysis was performed using SYBR Green 2X PCR Master Mix (Applied Biosystem). Each sample was run in triplicate and normalized to the expression of housekeeping beta-glucoronidase (GUSb) gene as previously described [6 (link)]. Primers are presented in Supplementary Table S1.