Limits...
Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

Castle J, Garrett-Engele P, Armour CD, Duenwald SJ, Loerch PM, Meyer MR, Schadt EE, Stoughton R, Parrish ML, Shoemaker DD, Johnson JM - Genome Biol. (2003)

Bottom Line: We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts.Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions.These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rosetta Inpharmatics, Merck & Co, Inc, 12040 115th Ave NE, Kirkland, Washington 98034, USA.

ABSTRACT
Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

Show MeSH

Related in: MedlinePlus

Intensity profile of RB1 exon edges in Jurkat cells. Median log intensities from 27 aligned exons show the edge profile for varying probe lengths (from 20 to 60 nucleotides) and formamide concentrations (see key). As the exons have different sizes, the average probe intensity within each exon is reported by a horizontal line between the two '0' points. The horizontal axis indicates the distance between the 5' end of the probe and the splicing acceptor site on the left side of the figure, and the distance between the 3' end of the probe and the splicing donor site on the right side of the figure. RB1 is expressed at low levels in the cell line K562; nevertheless, these edge profiles (data not shown) are similar but with lower intensities.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC328455&req=5

Figure 4: Intensity profile of RB1 exon edges in Jurkat cells. Median log intensities from 27 aligned exons show the edge profile for varying probe lengths (from 20 to 60 nucleotides) and formamide concentrations (see key). As the exons have different sizes, the average probe intensity within each exon is reported by a horizontal line between the two '0' points. The horizontal axis indicates the distance between the 5' end of the probe and the splicing acceptor site on the left side of the figure, and the distance between the 3' end of the probe and the splicing donor site on the right side of the figure. RB1 is expressed at low levels in the cell line K562; nevertheless, these edge profiles (data not shown) are similar but with lower intensities.

Mentions: Although longer 60-nucleotide 'exon probes' are better for detecting exons, shorter probes or higher formamide concentrations could be more accurate for pinpointing splice sites between exons and adjacent introns. To optimize detection of exon boundaries, we placed overlapping probes across all exon-intron boundaries at one-nucleotide intervals from 100 nucleotides within the intron to entirely within the exon, using the 27 exons of RB1 as a test case. We again varied probe length, testing 20-, 25-, 30-, 35-, 40-, 45-, 50-, and 60-nucleotide probes, as well as formamide concentrations, probe-step intervals, and analysis methods. Figure 3 shows the tiling intensity profiles for these 5' and 3' edge probes, using 35-nucleotide probes in 30% formamide. Figure 4 shows the averaged exon-intron edge profile at a range of formamide concentrations and probe lengths.


Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

Castle J, Garrett-Engele P, Armour CD, Duenwald SJ, Loerch PM, Meyer MR, Schadt EE, Stoughton R, Parrish ML, Shoemaker DD, Johnson JM - Genome Biol. (2003)

Intensity profile of RB1 exon edges in Jurkat cells. Median log intensities from 27 aligned exons show the edge profile for varying probe lengths (from 20 to 60 nucleotides) and formamide concentrations (see key). As the exons have different sizes, the average probe intensity within each exon is reported by a horizontal line between the two '0' points. The horizontal axis indicates the distance between the 5' end of the probe and the splicing acceptor site on the left side of the figure, and the distance between the 3' end of the probe and the splicing donor site on the right side of the figure. RB1 is expressed at low levels in the cell line K562; nevertheless, these edge profiles (data not shown) are similar but with lower intensities.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC328455&req=5

Figure 4: Intensity profile of RB1 exon edges in Jurkat cells. Median log intensities from 27 aligned exons show the edge profile for varying probe lengths (from 20 to 60 nucleotides) and formamide concentrations (see key). As the exons have different sizes, the average probe intensity within each exon is reported by a horizontal line between the two '0' points. The horizontal axis indicates the distance between the 5' end of the probe and the splicing acceptor site on the left side of the figure, and the distance between the 3' end of the probe and the splicing donor site on the right side of the figure. RB1 is expressed at low levels in the cell line K562; nevertheless, these edge profiles (data not shown) are similar but with lower intensities.
Mentions: Although longer 60-nucleotide 'exon probes' are better for detecting exons, shorter probes or higher formamide concentrations could be more accurate for pinpointing splice sites between exons and adjacent introns. To optimize detection of exon boundaries, we placed overlapping probes across all exon-intron boundaries at one-nucleotide intervals from 100 nucleotides within the intron to entirely within the exon, using the 27 exons of RB1 as a test case. We again varied probe length, testing 20-, 25-, 30-, 35-, 40-, 45-, 50-, and 60-nucleotide probes, as well as formamide concentrations, probe-step intervals, and analysis methods. Figure 3 shows the tiling intensity profiles for these 5' and 3' edge probes, using 35-nucleotide probes in 30% formamide. Figure 4 shows the averaged exon-intron edge profile at a range of formamide concentrations and probe lengths.

Bottom Line: We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts.Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions.These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rosetta Inpharmatics, Merck & Co, Inc, 12040 115th Ave NE, Kirkland, Washington 98034, USA.

ABSTRACT
Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

Show MeSH
Related in: MedlinePlus