Supplemental Data

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• Supplemental Data -

  Supplemental Information

  To further confirm the presence of the exon 1 sequence in the EG-VEGF transcript, a forward primer derived from this sequence was utilized in RT-PCR reactions. The expected 264 bp product was generated from d7 embryo, kidney and liver samples. No amplicon was generated from the ovary or testis samples (Supplemental Fig. 1A). Also, no EGVEGF amplicon is detected in reactions without reverse transcriptase (Supplemental Fig. 1B). Therefore, the EG-VEGF exon 1 sequence is present in d7 embryo, liver and kidney mRNA samples.

  Supplemental Experimental Procedures

  For RT-PCR, 2 µg of total RNA from d7 embryo, kidney, liver, ovary and testis was reverse transcribed for 2 hours at 42°C using 50 units MuLV reverse transcriptase, 1 uM oligo dT, RNase inhibitor, and dNTP Mix (all from Applied Biosystems). Controls included reactions without reverse transcriptase and reactions without RNA template.

  Subsequently, one tenth of each of these reactions was used for PCR using a two-step protocol: 95°C for 30 seconds and 72°C for 45 seconds, for 27 cycles. Reaction mix included 1 unit AmpliTaq Gold, PCR buffer II and dNTP mix. Oligo sequences were as follows: forward (5’-GTGTCTGACTGTGCTGTGATCACAGGG) and reverse (5’- GATGTTCTTC-AAGTCCATGGAGCAGC). The expected size of the product was 264 bp. One fifth of the PCR reactions was run on a 2% agarose gel containing ethidium bromide.

  Supplemental Figure 1

  EG-VEGF exon 1 sequence is present in the mRNA transcript. A. Photograph of an ethidium bromide stained gel containing RT-PCR reaction products. Contents of lanes 1-6 are as follows: no template, d7 embryo, kidney, liver, ovary and testis. The amplicon in lanes 2-4 is an expected 264 bp. B. Agarose gel with control reactions, generated without reverse transcriptase. Contents of the lanes, 1-5, are as follows: d7 embryo, kidney, liver, ovary, testis all without reverse transcriptase. Lane 6 is a positive control for the amplicon, one tenth of an RT-PCR reaction using liver as template. No amplicon is detected in lanes 1-5. GAPDH RT-PCR (data not shown), and Taqman (Fig. 4) reactions served as controls for RNA integrity. Size (bp) is indicated.