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MicroSAGE is highly representative and reproducible but reveals major differences in gene expression among samples obtained from similar tissues.

Blackshaw S, Kuo WP, Park PJ, Tsujikawa M, Gunnersen JM, Scott HS, Boon WM, Tan SS, Cepko CL - Genome Biol. (2003)

Bottom Line: Serial analysis of gene expression using small amounts of starting material (microSAGE) has not yet been conclusively shown to be representative, reproducible or accurate.We show that microSAGE is highly representative, reproducible and accurate, but that pronounced differences in gene expression are seen between tissue samples taken from different individuals.MicroSAGE is a reliable method of comprehensively profiling differences in gene expression among samples, but care should be taken in generalizing results obtained from libraries constructed from tissue obtained from different individuals and/or processed or stored differently.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute, 200 Longwood Avenue, Boston, MA 02115, USA.

ABSTRACT

Background: Serial analysis of gene expression using small amounts of starting material (microSAGE) has not yet been conclusively shown to be representative, reproducible or accurate.

Results: We show that microSAGE is highly representative, reproducible and accurate, but that pronounced differences in gene expression are seen between tissue samples taken from different individuals.

Conclusions: MicroSAGE is a reliable method of comprehensively profiling differences in gene expression among samples, but care should be taken in generalizing results obtained from libraries constructed from tissue obtained from different individuals and/or processed or stored differently.

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SAGE tag levels of cell-cycle regulatory genes as measured by SAGE. Tag levels are normalized to 55,000.
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Figure 3: SAGE tag levels of cell-cycle regulatory genes as measured by SAGE. Tag levels are normalized to 55,000.

Mentions: Additional analyses to determine the accuracy of microSAGE data were carried out by examining a series of libraries from mouse retina and hypothalamus. We have constructed SAGE libraries from mouse hypothalamus; total wild-type mouse retina at 2-day intervals from embryonic day (E) 12.5 through to postnatal day (P) 6.5; P10.5 wild-type mice along with littermates mutant for the crx gene (which encodes a Paired type homeodomain transcription factor that directly regulates transcription of many photoreceptor-enriched genes); adult mouse retina; and microdissected outer nuclear layer, which consists entirely of rod and cone photoreceptors [11]. We examined this very large dataset for expression of genes known to be photoreceptor specific (Figure 2). We found that photoreceptor-specific genes typically showing high, retina-specific expression that began around one week after birth, coinciding exactly with the previously reported increase in expression of rhodopsin and other rod-specific genes [13-15]. We typically saw higher expression of photoreceptor-specific genes in microSAGE libraries made from microdissected outer nuclear layer (97% rods versus 70% rods for whole retina) and, in many cases, higher expression in P10.5 crx+/+ samples relative to P10.5 crx-/- samples. These observations accurately reflect the known expression pattern of rod-specific genes [15]. Examining the temporal dataset for genes that are known to be selectively expressed in retinal progenitor cells (Figure 3), we found that high expression persisted until P2, followed by a rapid drop, with expression barely detectable in the adult. These data correlate well with the time course of mitosis within the developing retina [16,17].


MicroSAGE is highly representative and reproducible but reveals major differences in gene expression among samples obtained from similar tissues.

Blackshaw S, Kuo WP, Park PJ, Tsujikawa M, Gunnersen JM, Scott HS, Boon WM, Tan SS, Cepko CL - Genome Biol. (2003)

SAGE tag levels of cell-cycle regulatory genes as measured by SAGE. Tag levels are normalized to 55,000.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC153457&req=5

Figure 3: SAGE tag levels of cell-cycle regulatory genes as measured by SAGE. Tag levels are normalized to 55,000.
Mentions: Additional analyses to determine the accuracy of microSAGE data were carried out by examining a series of libraries from mouse retina and hypothalamus. We have constructed SAGE libraries from mouse hypothalamus; total wild-type mouse retina at 2-day intervals from embryonic day (E) 12.5 through to postnatal day (P) 6.5; P10.5 wild-type mice along with littermates mutant for the crx gene (which encodes a Paired type homeodomain transcription factor that directly regulates transcription of many photoreceptor-enriched genes); adult mouse retina; and microdissected outer nuclear layer, which consists entirely of rod and cone photoreceptors [11]. We examined this very large dataset for expression of genes known to be photoreceptor specific (Figure 2). We found that photoreceptor-specific genes typically showing high, retina-specific expression that began around one week after birth, coinciding exactly with the previously reported increase in expression of rhodopsin and other rod-specific genes [13-15]. We typically saw higher expression of photoreceptor-specific genes in microSAGE libraries made from microdissected outer nuclear layer (97% rods versus 70% rods for whole retina) and, in many cases, higher expression in P10.5 crx+/+ samples relative to P10.5 crx-/- samples. These observations accurately reflect the known expression pattern of rod-specific genes [15]. Examining the temporal dataset for genes that are known to be selectively expressed in retinal progenitor cells (Figure 3), we found that high expression persisted until P2, followed by a rapid drop, with expression barely detectable in the adult. These data correlate well with the time course of mitosis within the developing retina [16,17].

Bottom Line: Serial analysis of gene expression using small amounts of starting material (microSAGE) has not yet been conclusively shown to be representative, reproducible or accurate.We show that microSAGE is highly representative, reproducible and accurate, but that pronounced differences in gene expression are seen between tissue samples taken from different individuals.MicroSAGE is a reliable method of comprehensively profiling differences in gene expression among samples, but care should be taken in generalizing results obtained from libraries constructed from tissue obtained from different individuals and/or processed or stored differently.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute, 200 Longwood Avenue, Boston, MA 02115, USA.

ABSTRACT

Background: Serial analysis of gene expression using small amounts of starting material (microSAGE) has not yet been conclusively shown to be representative, reproducible or accurate.

Results: We show that microSAGE is highly representative, reproducible and accurate, but that pronounced differences in gene expression are seen between tissue samples taken from different individuals.

Conclusions: MicroSAGE is a reliable method of comprehensively profiling differences in gene expression among samples, but care should be taken in generalizing results obtained from libraries constructed from tissue obtained from different individuals and/or processed or stored differently.

Show MeSH