RT-PCR analysis of selected and MIR exons

Multiple alignment of mammalian interspersed repeat (MIR) exon in DMWD gene among mammals. Exon sequences are marked in blue, flanking intronic sequences are marked in black, and the canonical AG and GT dinucleotides at the 3'ss and 5'ss are marked in red. Nucleotide conservation is marked at the lower edge, with asterisks indicate full conservation and colons indicating partial conservation relative to the MIR consensus sequence (lower row). The divergence in percentage from the consensus MIR sequence is indicated under (MIR div); exon conservation in percentage compared with the human exon is indicated under (exon conserve); EST/cDNA accession confirming the exon insertion is indicated under (cDNA/EST holding evidence), and skipping is indicated under (cDNA/EST skipping evidence). Nonconserved nucleotides are marked in yellow. This panel is similar to panel a, except that the conservation is shown for the protein coding sequence. Total RNA was collected from SH-SY5Y human cell line and mouse brain tissue. Reverse transcription polymerase chain reaction (RT-PCR) analysis amplified the endogenous mRNA molecules using primers specific to the flanking exons. The PCR products were separated on an agarose gel, extracted and sequenced. A schema of the mRNA products is shown on the left and right. Columns 1 to 4 show the splicing pattern of orthologous human (H) and mouse (M) exons originating from the MIR element. Columns 1 and 2 show alternative splicing of an ortholog MIR element in both human and mouse, respectively (exon 4 in DMWD gene), and columns 3 and 4 show a constitutive pattern in both species (exon 5 in the MYT1L gene). Column 5 shows constitutive splicing of an element in the human exon 3 of FAM55C gene. All PCR products were confirmed by sequencing. We cannot fully reject the option that an exon that is constitutively spliced under the above conditions is alternatively spliced in other cells or conditions. However, the constitutive selection is also supported by EST/cDNA coverage.

Copyright information:

Taken from "Comparative analysis of transposed element insertion within human and mouse genomes reveals 's unique role in shaping the human transcriptome"

http://genomebiology.com/2007/8/6/R127

Genome Biology 2007;8(6):R127-R127.

Published online 27 Jun 2007

PMCID:PMC2394776.