Reduced allele specific annexin A5 mRNA levels in placentas carrying the M2/ANXA5 allele

a) Scheme of the N and M2 ANXA5 allelic differences on the DNA and mRNA level and amplification strategy from cDNA. utr Exon 1, non-translated Exon 1, tsp, transcription start point; *, nucleotide count start; 1., 2., 3., variant ANXA5 transcripts; forward primer G anneals to all N ANXA5 transcripts, forward primer A anneals to all M2 ANXA5 transcripts. b) N/M2 allele specific PCR does not generate product from N/N samples, as demonstrated by direct visualization on 1.5% agarose gel. Arrow points at the specific amplicon band of 131 bp in the N/M2 control lane, asterisks below the N/N lanes denote non-utilized primers. c) Melting curve analysis of N/M2 M2 allele specific products. Melting curves are homogenous at ca. 93 °C, indicative of a single amplicon. Base line non-specific products appear only in water samples at ca. 82 °C.

a) Expression ratio of ANXA5 N allele mRNA in heterozygous (N/M2) vs normal homozygous (N/N) placentas. Output was generated with the REST (2008) software. b) Expression ratio of ANXA5 M2 allele mRNA to N allele mRNA in heterozygous (N/M2) placentas. Output was generated with the 2^DDCt method. c) Typical western blot of placental protein samples. Blots were cut in strips around the relative molecular mass for α-tubulin (55 kDa) and annexin A5 (36 kDa). Lanes in upper rows were probed with anti-α-tubulin antibodies to ensure equal protein loading and lower row lanes were probed with anti-annexin A5 antibodies. Placental genotypes are indicated above each lane.