Table 1 Primer pairs used for PCR reactions GENE ACCESION NUMBER PRIMER SEQUENCE Tm MEIS1 [GenBank:NM_002398] F: CCC CAG CAC AGG TGA CGA TGA T R: TGC CCA TTC CAC TCA TAG GTC C 60 MEIS2 [GenBank:NM_170677] F: CCA TCG ACC TCG TCA TTG AT R: CCT CCT TTC TTC TGG CGT TTT T 60 PREP1 [GenBank:NM_004571] F: OICR-9429 cell line GGT TTT GGC CTG ATT CTA TTG C R: GTG GGG AGG GAG TGG TG 65 PREP2 [GenBank:NM_022062] F: GCC ACC AAT ATA ATG CGT TCT T R: GTG TTC CAA GCC CAG GTC 65 PBX1 [GenBank:NM_002585] F: CTA ACT CGC CCT CAA CTC C R: GTG TCC AGA TTG GCT GAA ATA G 60 PBX2 [GenBank:NM_002586] F: GGC GGC
TCT TTC AAT CTC TCA R: GTC TCG TTA GGG AGG GGA TGA C 65 PBX3 [GenBank:NM_006195] F: CAA GGG TCC CAA GTC GG R: TGG CCT AAT TGG ATA AAG TGC T 60 PBX4 [GenBank:NM_025245] F: ATG GGG AAG TTT CAA GAA GAG G R: ATC TCG AGT CGC AGC AGA C 65 GAPDH [GenBank:NM_002046] F: CAC TGC CAC CCA GAA GAC TGT G R: TGT AGG CCA TGA GGT CCA CCA C 60 RPL32 [GenBank:NM_000994] F: GCA TTG ACA ACA GGG TTC GTA G R: ATT TAA ACA GAA AAC GTG CAC A 60 ACTB [GenBank:NM_001101] F: TCC GCA AAG ACC TGT ACG R: AAG AAA GGG TGT AAC GCA ACT A 60 Figure 1 Analysis of primers used for amplification of Three-amino-acid loop-extension (TALE) family member genes. A) A pull of cDNA Temsirolimus obtained from leukemia-derived cell lines was utilized to test the specificity and efficiency of each set of primers in the amplification of TALE family genes. After 40
cycles of amplification by conventional PCR, the PCR products were separated into 2% agarose gels and visualized under Ultraviolet (UV) light. Amplification
products of the reference genes employed (RPL32 and ACTB) are also included. LY2603618 clinical trial The 1 Kb Plus DNA Ladder (Invitrogen, Life Science) is shown in the left line; B) Amplification of PBX1 in leukemia-derived cell lines and in healthy controls separated into 2% agarose gels and visualized under UV light (upper panel). Genome map of complete (a) and alternative (b) splicing of PBX1; C) Sequence of alternative splicing of PBX1 showing adjacent coding regions of the deleted exon. Next, we proceeded to analyze the expression of Thiamet G TALE genes by qRT-PCR in leukemia-derived cell lines. We employed five cell lines including Jurkat, CEM, MOLT-4, K562, and HL-60; the first three are lymphoblastic, and the latter two, myeloid. We determined the crossing point for each target gene and subsequently normalized this with the crossing point of an internal reference gene to calculate the ΔCP, which represents an absolute and more comparative value (see Materials and Methods). It is important to bear in mind that the ΔCP value is inversely proportional to gene expression. To obtain more consistent results, we use two different reference genes: RPL32, and ACTB. As can be observed in Figure 2, results obtained with RPL32 and ACTB follow the same tendency. In this regard, RPL32 and ACTB were selected as confident reference genes.
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