Table 1. Primers and probes used for PCR analysis.
PCR | Gene sequence | Name | 5′- 3′ | Annealing temperature (°C) | Cycles | Product size (bp) | Position (nt) |
Qualitative | BCR-JAK2 | BCR-Fw-T | GCCATGGTGGACCCGGTGG | 55 | 35 | 2260 | 594–6121 |
JAK2-Rv-T | AAGGTCATTTCTTTCATCCAGCC | 3884–39062 | |||||
Quantitative | BCR-JAK2 | FRET-probe-FL | GTCTTGCGGACGCCCACGA-FL * | 1211–12291 | |||
FRET-probe-LC640 | LC640-GGTGGCCTCGGACACGACAACC+ | 1188–12091 | |||||
BCR-B-Fw | CCCCCGGAGTTTTGAGGATTG | 63 | 45 | 997 | 1547–15671 | ||
JAK2-3-Rv | GGCCACAGAAAACTTGCTCTC | 3576–35962 | |||||
BCR | BCR-B-Fw | CCCCCGGAGTTTTGAGGATTG | 63 | 45 | 301 | 1547–15671 | |
BCR-B-Rv | ATCGTTGGGCCAGATCTGCC | 1828–18471 | |||||
Bcl-xL | Bcl-xL Fw | TCAGAGCTTTGAGCAGGTAGTG | 58 | 45 | 187 | 726–7473 552–5734 | |
Bcl-xL Rv | TCCCGTAGAGATCCACAAAAG | 935–9553 761–7814 | |||||
Osm | Osm Fw | AGAATCAGGCGAACCTCACGG | 58 | 45 | 74 | 217–237 5 | |
Osm Rv | GTGTGTTCAGGTTTTGGAGGC | 271–2915 | |||||
Gapdh | Gapdh Fw | AGAAGGTGGTGAAGCAGGCATC | 58 | 45 | 116 | 820–8416 | |
Gapdh Rv | CGGCATCGAAGGTGGAAGAGTG | 915–9366 | |||||
H3 | H3 Fw | AAAGCCGCTCGCAAGAGTGCG | 62 | 35 | 201 | 185–2057 | |
H3 Rv | ACTTGCCTCCTGCAAAGCAC | 387–4067 |
Fw, forward; Rv, reverse; FL, fluorescein; LC640, LightCycler Red.
*,+: inverse and complementary probe sequences to human BCR;
human BCR (NM_004327);
human JAK2 (NM_ 004972);
human Bcl-xL (BCLXL or BCL2L1) (NM_ 1385781);
mouse Bcl-xL (BCL2-like 1 or Bcl2l1) (NM_ 009743) and
mouse Osm (oncostatin M) (NM_001013365.2) [39];
mouse Gapdh (glyceraldehyde-3-phosphate dehydrogenase) NM_008084.2),
human H3 (H3F3A) (NM_002107.3).
Primers for Bcl-xL and H3F3A are designed to hybridize with both human and mouse sequences (patient samples and Ba/F3 cells, respectively).