Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis
Total RNA was extracted from cultured cells using RNAeasy kit (Quiagen). Aliquots (1 µg) of total RNA from the cells were reverse transcribed in the presence of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 µM dNTPs, and 0.5 µg oligo-dT(12–18) with 200 U Superscript RNase H-Reverse Transcriptase (Invitrogen). PCR amplification was performed using standard procedure with Taq Polymerase. Aliquots of cDNA equivalent to 50 ng of total RNA were amplified in 25 µl reactions containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , 50 pmol of each primer, 400 µM dNTPs, and 0.5 U AmpliTaq DNA polymerase (Perkin-Elmer). PCR was performed using the following thermal profile: 4 min at 94°C; 1 min at 94°C, 1 min at 60°C, 1.5 min at 72°C, for 30–40 cycles; 7 min at 72°C, and finally a soak at 4°C overnight. The following day, 10 µl aliquots of the amplified products were run on a 2% agarose Tris–acetate gel containing 0.5 mg/ml ethidium bromide. The products were visualized through a UV transilluminator, captured in a digital format using Quantify One Gel Analysis software (Bio-Rad Laboratories) on a Macintosh G4 computer.
The PCR primers specific to each transcript were as follows: GFAP, forward (F), 5′-TCATCGCTCAGGAGGTCCTT–3′ Reverse (R), 5′-CTG TTGCCAGAGATGGAGGTT–3′; MAP2 (F) 5′-GAAGACTCGCATCCGAATGG–3′, (R) 5′-CGCAGGATAGGAGGAAGAGACT–3′; MBP (F) 5′-TTAGCTGAATTC GCGTGTGG–3′, (R) 5′-GAGGAAGTGAATGAGCCGGTTA-3′ were deigned using the Primer Designer software, Version 2.0 (Scientific and Educational Software) [48]. 18S, β-tubulin class III, N-CAM, Nestin, NF-M, Notch-1 primers [51]. Oct4, Nanog primers [11]. FOXa2 (HNF3B), Brachyury primers [52].