Abstract
Using gene arrays, it is currently possible to simultaneously measure mRNA levels of many genes in any tissue of interest. Undoubtedly, comprehensive measurements of gene expression as part of carefully designed experiments will continue to further our understanding of audition and have the potential to open up new avenues of research. This chapter describes a reliable protocol to prepare high-quality biotin-labeled RNA target, specifically for oligonucleotide array experiments. The procedure includes isolation of high-quality total RNA, synthesis of double-stranded cDNA engineered for in vitro transcription with T7 RNA polymerase, subsequent in vitro transcription in the presence of biotin-labeled ribonucleotides, and fractionation of the RNA to ≈500 bp fragments, suitable for oligonucleotide array experiments. Because the membranous labyrinth is composed of functionally interdependent cellular structures, which themselves contain numerous, highly differentiated cell types, comprehensive analysis of gene expression in the cochlea is best complemented by immunohistotochemical studies or, if no suitable antibodies are available, by in situ hybridization studies. Either one of these techniques will identify the specific cell types that express the genes of interests.
| Original language | English (US) |
|---|---|
| Title of host publication | Methods in Molecular Biology |
| Publisher | Humana Press |
| Pages | 21-29 |
| Number of pages | 9 |
| Volume | 493 |
| ISBN (Print) | 9781934115626 |
| DOIs | |
| State | Published - 2009 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 493 |
| ISSN (Print) | 10643745 |
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Keywords
- Cochlea
- Gene chips
- Gene expression
- Membranous labyrinth
- Oligonucleotide microarrays
ASJC Scopus subject areas
- Molecular Biology
- Genetics
Cite this
Synthesis of biotin-labeled RNA for gene expression measurements using oligonucleotide arrays. / Vázquez, Ana E.; Nie, Liping; Yamoah, Ebenezer N.
Methods in Molecular Biology. Vol. 493 Humana Press, 2009. p. 21-29 (Methods in Molecular Biology; Vol. 493).Research output: Chapter in Book/Report/Conference proceeding › Chapter
}
TY - CHAP
T1 - Synthesis of biotin-labeled RNA for gene expression measurements using oligonucleotide arrays
AU - Vázquez, Ana E.
AU - Nie, Liping
AU - Yamoah, Ebenezer N.
PY - 2009
Y1 - 2009
N2 - Using gene arrays, it is currently possible to simultaneously measure mRNA levels of many genes in any tissue of interest. Undoubtedly, comprehensive measurements of gene expression as part of carefully designed experiments will continue to further our understanding of audition and have the potential to open up new avenues of research. This chapter describes a reliable protocol to prepare high-quality biotin-labeled RNA target, specifically for oligonucleotide array experiments. The procedure includes isolation of high-quality total RNA, synthesis of double-stranded cDNA engineered for in vitro transcription with T7 RNA polymerase, subsequent in vitro transcription in the presence of biotin-labeled ribonucleotides, and fractionation of the RNA to ≈500 bp fragments, suitable for oligonucleotide array experiments. Because the membranous labyrinth is composed of functionally interdependent cellular structures, which themselves contain numerous, highly differentiated cell types, comprehensive analysis of gene expression in the cochlea is best complemented by immunohistotochemical studies or, if no suitable antibodies are available, by in situ hybridization studies. Either one of these techniques will identify the specific cell types that express the genes of interests.
AB - Using gene arrays, it is currently possible to simultaneously measure mRNA levels of many genes in any tissue of interest. Undoubtedly, comprehensive measurements of gene expression as part of carefully designed experiments will continue to further our understanding of audition and have the potential to open up new avenues of research. This chapter describes a reliable protocol to prepare high-quality biotin-labeled RNA target, specifically for oligonucleotide array experiments. The procedure includes isolation of high-quality total RNA, synthesis of double-stranded cDNA engineered for in vitro transcription with T7 RNA polymerase, subsequent in vitro transcription in the presence of biotin-labeled ribonucleotides, and fractionation of the RNA to ≈500 bp fragments, suitable for oligonucleotide array experiments. Because the membranous labyrinth is composed of functionally interdependent cellular structures, which themselves contain numerous, highly differentiated cell types, comprehensive analysis of gene expression in the cochlea is best complemented by immunohistotochemical studies or, if no suitable antibodies are available, by in situ hybridization studies. Either one of these techniques will identify the specific cell types that express the genes of interests.
KW - Cochlea
KW - Gene chips
KW - Gene expression
KW - Membranous labyrinth
KW - Oligonucleotide microarrays
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UR - http://www.scopus.com/inward/citedby.url?scp=84934436295&partnerID=8YFLogxK
U2 - 10.1007/978-1-59745-523-7_2
DO - 10.1007/978-1-59745-523-7_2
M3 - Chapter
AN - SCOPUS:84934436295
SN - 9781934115626
VL - 493
T3 - Methods in Molecular Biology
SP - 21
EP - 29
BT - Methods in Molecular Biology
PB - Humana Press
ER -