Ssofast evagreen reagent

Total RNA was isolated from 5 × 10⁶ cells using TRIzol^® reagent (Invitrogen, Paisley, UK), according to the manufacturer's instructions. Complementary DNA (cDNA) was transcribed using SuperScript II Reverse Transcriptase (Invitrogen, Paisley, UK), starting from 1 μg/μl of high pure RNA and samples were tested in triplicate using the SsoFast EvaGreen reagents (Bio-Rad).
Primer sets were the following:
MICAFw–GACTTGACAGGGAACGGAAA, MICARev–CAGGTTTTGGGAGAGGAAGA; MICBFw–CAGCTACTGGGTCCACTGGT, MICBRev–GTTGGTCATGATCCCTTTGC; CB1Fw–CTTCACGGTCCTGGAGAACC, CB1Rev–ACCCCACCCAGTTTGAACAG and the house keeping gene was the β2-microglobulin: β2Fw-CCTGGATTGCTATGTGTCTGG, β2Rev–GGAGCAACCTGCTCAGATACA or β-actin: ActinFw–CACTGTGCCCATCTACGAGG, ActinRev–TGGCCATCTCTTGCTCGAAG; ULBP1Fw-CGGTGCTAATGGATGGAACT, ULBP1Rev-TGGTCAGTGCATCAAAAGGA; ULBP2Fw-TCAAACTCGCCCTTCTGTCT, ULPB2Rev-GTGCAGGAATTCCATCAGGT; ULBP3Fw–CTGGGGAAAACAACTGGAAA, ULBP3Rev-ATCGAAGCTGAACTGCCAAG; ULBP4Fw–CCCTGACTTCTAGCCCTGTG, ULBP4Rev-AGAAGACCTGCGCTTCACAC; GAPDHFw–CGCTCTCTGCTCCTCCTGTTC, GAPDHRev-TTGACTCCGACCTTCACCTTCC.
qRT-PCR protocol was: pre-heating step for 3 minutes at 95°C, then 40 cycles at 95°C for 10 seconds and 60° for 30 seconds and last end-step at 65°C for 10 seconds. Finally, results were analyzed with 2^−ßßCt method.

Total RNA was isolated using the NucleoSpin RNA II kit (Macherey-Nagel, Mountain View, CA, USA). Complementary DNA (cDNA) was transcribed using SuperScript II Reverse Transcriptase (Invitrogen), starting from 1 μg of high pure RNA. RAD51 gene expression profile was evaluated with specific primer sets and using SsoFast EvaGreen reagents(Bio-Rad). RAD51 primers (PrimePCR™ PreAmp for SYBR® Green Assay: RAD51, Human Bio-Rad, qHsaCED00421333); β2-microglobulin (primers: Fw, CCTGAATTGCTATGTGTCTGGG, Rev, AATGCGGCATCTTCAAACCTC) was used as housekeeping gene. qRT-PCR protocol was: a pre-heating step for 3 min at 95°C, 40 cycles at 95°C for 10 s and 60° for 30 s and last end-step at 65°C for 10 s. Results were analyzed with 2^−ddCt method (15 (link)).

Neurospheres treated or not with 8µM GW6471 in normoxia and with 8-16µM in hypoxia were harvested with Trizol (Invitrogen) and total RNA was isolated using the Nucleo Spin RNA II kit (Macherey-Nagel) according manufacturer’s instructions. cDNA was transcribed using Super Script II Reverse Transcriptase (Invitrogen), starting from 0.5 micrograms of high pure RNA. Mevalonate genes expression profiles were evaluated with specific primer sets (Table 1) and using Sso Fast Eva Green reagents (Bio-Rad), β2-microglobulin was used as housekeeping gene. qRT-PCR protocol was: a pre-heating step for 3 min, at 95°C, 40 cycles at 95°C for 10 seconds and 60°C for 30 seconds and last end-step at 65°C for 10 seconds. Results were analyzed with 2-^ΔΔCt method [68 (link)].