Screening of cell lines for
SET/NUP214 mRNA expression was performed applying RT-PCR. RNA was prepared using the Trizol reagent (Invitrogen, Karlsruhe, Germany). For mRNA quantification, reverse transcription was performed using the SuperScript II reverse transcriptase kit (Invitrogen, Karsruhe, Germany). Previous studies identified
SET exon 7/
NUP214 exon 17 and
SET exon 7/
NUP214 exon 18 fusions in T-ALL and AML patients [
4,
5,
10]. We applied primers from
SET exon 6 and
NUP214 exon 20 for
SET-NUP214 expression screening. Analyses were repeated with previously described primers from
SET exon 7 and
NUP214 exon 18 [
10]:
SET exon 6 forward: 5'-GAA GAG GCA GCA TGA GGA AC-3';
NUP214 exon 20 reverse: 5'-TAC TTT GGG CAA GGA TTT GG-3';
SET exon 7 forward: 5'-TGA CGA AGA AGG GGA TGA GGA T-3';
NUP214 exon 18 reverse: 5'-ATC ATT CAC ATC TTG GAC AGC A-3'. The same
NUP214 exon 18 reverse primer was used in combination with alternative exon 1 forward primers to detect
TAF-Iα-NUP214 and
TAF-Iβ (
SET)-
NUP214 mRNA isoforms:
TAF-Iα exon 1 forward: 5'-TAA ACG CCA GTC TCC ACT CC-3',
TAF-Iβ (
SET) exon 1 forward: 5'-AGC TCA ACT CCA ACC ACG AC-3'. For the determination of genomic
SET and
NUP214 breakpoints in cell lines LOUCY and MEGAL, genomic PCR was performed with the following sets of primers: (i)
SET exon 7 forward: 5'-TGA CGA AGA AGG GGA TGA GGA T-3';
NUP214 exon 18 reverse: 5'-ATC ATT CAC ATC TTG GAC AGC A-3'. (ii)
SET intron 8/exon 8 forward: 5'-TCA GGA GGA TGA AGG AGA AGA-3';
NUP214 intron 17/18 reverse: 5'-GAG GTG GCA GAG AGG TGG TA-3'; (iii)
SET exon 8 forward: 5'-CTG CCA CTC AAT GGG AGA AT-3';
NUP214 intron 17/18 reverse: 5'-ACA AGA ATT ACC CGG GTG TG-3'; PCR was performed in a total volume of 50 μl with a DNA thermal cycler (Perkin Elmer Cetus, Heidelberg, Germany) for 35 cycles under standard conditions. Products were electrophoresed in 1.2% agarose gels and observed under UV light. PCR products were ligated into the pGEM-T Easy Vector System (Promega, Mannheim, Germany) and sequenced (Eurofins MWG Operon, Martinsried, Germany).