Seriai analysis of gene expression: rapid RT-PCR analysis of unknown SAGE tags

Berg, Anke van den; Leij, Judith van der; Poppema, Sibrand

doi:10.1093/nar/27.17.e17-i

Cited by 20 publications

(5 citation statements)

References 10 publications

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“…This fragment in turn could then successfully be used for BLAST search and annotation of the tags. Previously, several PCR techniques were reported to recover cDNA fragment from 13-to 15-bp SAGE tags (23,24). However, it appeared always difficult to determine the appropriate conditions for a specific amplification of cDNAs from each gene because the SAGE tag primers were too short.…”

Section: Discussionmentioning

confidence: 99%

Gene expression analysis of plant host–pathogen interactions by SuperSAGE

Matsumura

Reich

Ito

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

266

194

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The type III restriction endonuclease EcoP15I was used in isolating fragments of 26 bp from defined positions of cDNAs. We call this substantially improved variant to the conventional serial analysis of gene expression (SAGE) procedure ''SuperSAGE.'' By applying Super-SAGE to Magnaporthe grisea (blast)-infected rice leaves, gene expression profiles of both the rice host and blast fungus were simultaneously monitored by making use of the fully sequenced genomes of both organisms, revealing that the hydrophobin gene is the most actively transcribed M. grisea gene in blast-infected rice leaves. Moreover, SuperSAGE was applied to study gene expression changes before the so-called hypersensitive response in INF1 elicitor-treated Nicotiana benthamiana, a ''nonmodel'' organism for which no DNA database is available. Again, SuperSAGE allowed rapid identification of genes up-or down-regulated by the elicitor. Surprisingly, many of the down-regulated genes coded for proteins involved in photosynthesis. SuperSAGE will be especially useful for transcriptome profiling of two or more interacting organisms like hosts and pathogens, and of organisms, for which no DNA database is available.

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Section: Discussionmentioning

confidence: 99%

Gene expression analysis of plant host–pathogen interactions by SuperSAGE

Matsumura

Reich

Ito

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

266

194

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“…To achieve this goal, two sets of techniques were developed. First, two specific PCR-based techniques were designed, termed "rapid analysis of unknown SAGE tags" (RAST, [47]) and "generation of longer cDNA fragments from SAGE tags for gene identification" (GLGI, [48]; see an updated protocol at [49]). Though technologically different, both protocols share the same principle: an unknown nucleotide sequence located distal (3'-) from the SAGE tag of interest is amplified using (i) an extended sequence containing the SAGE tag and (ii) Poly(A)-derived sequence as the primers.…”

Section: Further Development Of Sage Techno-logymentioning

confidence: 99%

Serial Analysis of Gene Expression (SAGE): 13 Years of Application in Research

Anisimov¹

2008

CPB

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A number of molecular methods of gene expression analysis can approach genomic level. Among those, Serial Analysis of Gene Expression (SAGE) stands out. Unlike many other techniques, SAGE allows both qualitative and quantitative analysis of previously unknown transcripts. Over the course of the last 13 years, SAGE has became a recognized tool of large-scale gene expression profiling, being used extensively in human, animal, yeast and plant studies of various nature. A number of important adaptations was introduced both to the protocol of SAGE library construction and to the analytical algorithm employed. Moreover, some variations of the original protocol (MAGE, SADE, microSAGE, miniSAGE, longSAGE, superSAGE, deepSAGE, etc.) were derived to improve the utility of SAGE in certain conditions. Current review aims comparing the benefits and drawbacks of the techniques for high-throughput gene expression analysis (including SAGE) in a realistic, balanced manner. Issues related to modifications to the original protocol and further development of the SAGE are discussed.

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“…The conditions used for rapid reverse transcription-PCR analysis of unknown SAGE tags followed the procedures described in ref. 13. corresponding to a SAGE tag may provide misleading information.…”

Section: Identifying the Correct Sequence From Multiple Sequences Thatmentioning

confidence: 99%

“…Direct amplification of the specific template with our strategy will be very useful for confirmation of the validity of a particular SAGE tag. During the course of our research, we became aware of a report describing a method of rapid reverse transcription-PCR analysis of unknown SAGE tags (13). The authors used sense primers that were designed based on SAGE tags.…”

Section: Identifying the Correct Sequence From Multiple Sequences Thatmentioning

confidence: 99%

Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification

Chen

Rowley²,

Wang³

2000

Proc. Natl. Acad. Sci. U.S.A.

104

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We have developed a technique called the generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI), to convert SAGE tags of 10 bases into their corresponding 3 cDNA fragments covering hundred bases. A primer containing the 10-base SAGE tag is used as the sense primer, and a single base anchored oligo(dT) primer is used as an antisense primer in PCR, together with Pfu DNA polymerase. By using this approach, a cDNA fragment extending from the SAGE tag toward the 3 end of the corresponding sequence can be generated. Application of the GLGI technique can solve two critical issues in applying the SAGE technique: one is that a longer fragment corresponding to a SAGE tag, which has no match in databases, can be generated for further studies; the other is that the specific fragment corresponding to a SAGE tag can be identified from multiple sequences that match the same SAGE tag. The development of the GLGI method provides several potential applications. First, it provides a strategy for even wider application of the SAGE technique for quantitative analysis of global gene expression. Second, a combined application of SAGE͞GLGI can be used to complete the catalogue of the expressed genes in human and in other eukaryotic species. Third, it can be used to identify the 3 cDNA sequence from any exon within a gene. It can also be used to confirm the reality of exons predicted by bioinformatic tools in genomic sequences. Fourth, a combined application of SAGE͞GLGI can be applied to define the 3 boundary of expressed genes in the genomic sequences in human and in other eukaryotic genomes.

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Seriai analysis of gene expression: rapid RT-PCR analysis of unknown SAGE tags

Cited by 20 publications

References 10 publications

Gene expression analysis of plant host–pathogen interactions by SuperSAGE

Gene expression analysis of plant host–pathogen interactions by SuperSAGE

Serial Analysis of Gene Expression (SAGE): 13 Years of Application in Research

Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification

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