Quantitative RT-PCR analysis was performed according to the procedures described by Kanaoka et al.53 (link). At DIV 7 and 14, total RNA was extracted from co-cultured cells using the ISOSPIN Cell & Tissue RNA (Nippon Gene, Tokyo, Japan). Quantitative PCR was conducted on a LightCycler Nano System (Roche Diagnostics, Mannheim, Germany) using primers (see Table 1 ). Analysis of PCR data was performed using the comparative Ct method.
Oligonucleotide sequences of quantitative RT-PCR primer sets.
| Primer | Sequence (5′-3′) |
|---|---|
| (Forward) | |
| (Reverse) | |
| PTPRD-F | 5′-CCC CCA GGT TTA CAC GAA CTC-3′ |
| PTPRD-R | 5′-ATC CAG ACC CAT CGT CAA ATT C-3′ |
| Sema4D-F | 5′-CCT TGA GGA CGG AGT ATG CC-3′ |
| Sema4D-R | 5′-TCT GGA TCA CGT CAG CAA AGA-3′ |
| PlxnB1-F | 5′-CAC ACA TCT ACT ACA CTT GGC AA-3′ |
| PlxnB1-R | 5′-CAA TCC CGG CTG TCA TTC AC-3′ |
| Slitrk3-F | 5′-TGA AGC CAA GCA TAG CTG AAA-3′ |
| Slitrk3-R | 5′-ATC AGG GGA ATT GGG GTA GTC-3′ |
| Cntn5-F | 5′-ACT CCT CAG ATG CCT TCA GAC A-3′ |
| Cntn5-R | 5′-AGT TCC ATT CCG AAG CCA TCT G-3′ |
| Caspr4-F | 5′-TTT GGA ACG CAG CTT CCT TTA-3′ |
| Caspr4-R | 5′-GAG AGG GCT GTC GTC TTG AAA-3′ |
| GFAP-F | 5′-GCA AAA GCA CCA AAG AAG GGG A-3′ |
| GFAP-R | 5′-ACA TGG TTC AGT CCC TTA GAG G-3′ |
| MAP2-F | 5′-GCC AGC CTC AGA ACA AAC AG-3′ |
| MAP2-R | 5′-AAG GTC TTG GGA GGG AAG AAC-3′ |
| Tubb3-F | 5′-TAG ACC CCA GCG GCA ACT AT-3′ |
| Tubb3-R | 5′-GTT CCA GGT TCC AAG TCC ACC-3′ |
| β-actin-F | 5′-GGC TGT ATT CCC CTC CAT CG-3′ |
| β-actin-R | 5′-CCA GTT GGT AAC AAT GCC ATG T-3′ |