Quantifast sybr green

Total RNA was purified from A375M6 cells using the RNeasy mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The reverse transcription reaction was performed with QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) using 1 μg of total RNA. mRNA expression was performed using QuantiFast SYBR Green (Qiagen, Hilden, Germany). The primers used were: NANOG: 5′-ACCTTGGCTGCCGTCTCTGG-3′ (forward), 5′-AGCAAAGCCTCCCAATCCCAAACA-3′ (reverse); KLF4: 5′-GCAGCCACCTGGCGAGTC TG-3′ (forward), 5′-CCGCCAGCGGTTATTCGGGG-3′ (reverse); OCT3/4: 5′-TTTTGGTACCCCAGGCTATG-3′ (forward), 5′-GCAGGCACCTCAGTTTGAAT-3′ (reverse) and CD271: 5′-CGAGGCACCACCGACAACCT-3′ (forward), 5′-TGGTTCACTGGCCGGCTGTT-3′ (reverse). β2 was used as normalizer. All the amplifications were run on 7500 Fast Real-Time PCR System (BioRad, Hercules, CA, USA). Data were reported as relative quantity with respect to the calibrator sample using the −ΔΔ 2 Ct method.

Neurospheres were collected before plating on PLL (undifferentiated NSCs) or after differentiation for 1 or 4 days. RNA was extracted using the RNeasy Micro Kit (Qiagen) and quantified with a Nanodrop-1000 Spectrophotometer (NanoDrop Technologies, Inc). qRT-PCR was performed as described in ref. 36 (link). Complementary DNA was analyzed by qRT-PCR (5 ng RNA equivalent/reaction) using 0.2 µM target-specific primers and QuantiFast SYBR Green (Qiagen). Primers were validated by qRT-PCR analysis on serial dilutions of a positive control complementary DNA to assess their specificity and efficiency. Forty qRT-PCR cycles were performed on 7900HT instrument (Applied Biosystems) equipped with the Sequence Detection Systems (SDS) 2.3 software. Data were normalized using the expression of the housekeeping genes Actin and HPRT1 as in ref. 36 (link). C_t values were converted into fold-expression values relative to the control using qBase^PLUS software (Biogazelle).

RD cells were seeded at 1 × 10^∧5 cells per well in 24-well plates and subsequently infected with the wild-type (WT) E7 or E7 R2 mutants at a multiplicity of infection (MOI) of 0.01 or 1000 E7 RNA copies per cell. One hour post infection the inoculum was removed and cells were washed with phosphate buffered saline (PBS) before adding 500 μl cell culture medium. At the indicated times post infection the cell culture medium was aspirated and stored at −80°C. Where applicable, cells were lysed in 300 μl RLT lysis buffer and stored at −80°C before RNA isolation with the RNease kit (Qiagen). Viral titres were determined in an end point dilution assay (EPDA) by determining the tissue culture infectious dose 50% (TCID50) in RD cells.
Total RNA was harvested according to the manufacturer’s protocol (RNeasy, Qiagen). In a one-step reaction Quantifast Sybr green (Qiagen) total RNA was reverse transcribed with gene specific primers amplifying either E7 (primer pair E7 5’-UTR) or the internal control GAPDH (Supplementary file 1A) using the Stepone plus cycler (Applied Biosystems).