Journal: PLoS ONE
Article Title: Atlas of tissue- and developmental stage specific gene expression for the bovine insulin-like growth factor (IGF) system
Figure Lengend Snippet: Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants. The exon-intron structure of bovine insulin/insulin-like growth factor 2 ( INS/IGF2 , GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters ( IGF2 -P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human IGF2 -P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine IGF2 -P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 ( IGF2 -P1e2) and exon 3 ( IGF2 -P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for IGF2 -P1e2) and exons 3 and 8 (for IGF2 -P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since IGF2 -transcript P1 exon 2 is part of the first exonic region of transcript IGF2 -P0, and exon 3 is present in both IGF2- P0 and IGF2- P1 transcripts, the IGF2 -P1e2 and -P1e3 amplicons could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from IGF2- P2 promoter which comprise leading exon 4 ( IGF2 -P2e4) or leading exons 4 and 5 ( IGF2 -P2e5) as well as the protein coding exons. The forward primer for IGF2 -P2e4 was designed to span the junction of exons 4 and 8, and for IGF2 -P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify IGF2 -P3 and IGF2 -P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in S3 Table .
Article Snippet: Target specificity and integrity was confirmed via sequencing of selected amplicons on a 3730xl DNA Analyzer (Applied Biosystems, Inc., Foster City, CA, USA), plots of the melting curve derived by Mastercycler® ep Realplex software (Eppendorf, Inc., Hamburg, Germany), and by electrophoresis of PCR products on a 2% agarose gel (Agarose low EEO, AppliChem GmbH, Darmstadt, Germany) and visual inspection under UV with Gel DocTM 1000 Single Wavelength Mini-Transilluminator, using Quantity One image analyzing software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) after staining with GelRed™ Nucleic Acid Stain (Biotium, Inc., Hayward, CA, USA).
Techniques: Amplification, Variant Assay, Sequencing, Derivative Assay, Polymerase Chain Reaction