2″,”term_id”:”14589887″,”term_text”:”NM_004360.2″}}NM_004360.2 (CDH1), respectively, where www.selleckchem.com/products/PD-0332991.html +1 corresponds to the A of the ATG translation initiation codon. SMAD4 transcript analysis Fresh venous blood samples (2.5 ml) were collected into PAXgene Blood RNA tubes (PreAnalytiX; Qiagen, Hilden, Germany) containing RNA stabilising solution. Total RNA was extracted by use of the PAXgene Blood RNA Kit (Qiagen) according to the manufacturer’s protocol. First strand cDNA was synthesised from 2�C3 ��g of total RNA by random hexamer�\primed reverse transcription with the Superscript First Strand System for RT�\PCR (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer’s protocol. Reverse transcriptase PCR (RT�\PCR) fragments were obtained according to a standard PCR protocol, using a forward primer localised in exon 6 and a reverse primer in exon 10.
RT�\PCR products were separated on a 2% agarose gel and visualised with ethidium bromide on an ultraviolet imaging system (Biorad, San Diego, California, USA). Individual bands were excised from the gel and eluted with the High Pure PCR Product Purification Kit (Roche, Basel, Switzerland). Eluted DNA was reamplified with the same pair of primers and sequenced as described above. Detection of large genomic deletions by MLPA Large deletions or duplications were searched for with the MLPA assay for JPS. The newly developed MLPA Test Kit (SALSA P158�\JPS; MRC�\Holland, Amsterdam, The Netherlands) contains 15 paired probes from the SMAD4 region (1 probe for the promoter region, 3 probes for the first two noncoding exons and 11 probes for the coding exons), and 17 probes from the BMPR1A region (3 probes for the first 2 noncoding exons and 14 probes for 10 of the 11 coding exons).
No probe could be designed for coding exon 5 of the BMPR1A gene because of its high homology to the BMPR1A pseudogene. The MLPA kit also contains nine probes for the coding region of the PTEN gene. Deletion screening was performed according to the manufacturer’s protocol. Briefly, 75 ng of genomic DNA in 5 ��l TE buffer was heat�\denaturated for 5 min at 98��C and hybridised overnight (16 h) at 60��C with the set of MLPA probes. Next, hybridised products were ligated (at 54��C) and amplified by PCR (30 cycles), and PCR fragments were separated on an ABI 3100 capillary sequencer. Data were analysed using GeneMapper V.4.0 software (Applied Biosystems, Darmstadt, GSK-3 Germany) and gene dosage was calculated using the Coffalyser V4 program (MRC�\Holland). All identified deletions were confirmed in a second independent reaction. Where possible, segregation of the deletions with the disease in the families was examined.