An antibody-based microarray assay for small RNA detection.

Nucleic Acids Research
Zonglin HuStephen H Leppla

Abstract

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 microl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.

References

May 1, 1986·Journal of Immunological Methods·S J BoguslawskiR J Carrico
Mar 1, 1987·Analytical Biochemistry·B D Stollar, A Rashtchian
Jun 24, 2000·Nature·D J Lockhart, E A Winzeler
Jul 11, 2001·Genes & Development·K M WassarmanS Gottesman
Jan 24, 2002·Molecular Cell·Thorleif MøllerPoul Valentin-Hansen
Jul 16, 2002·Annual Review of Biomedical Engineering·Michael J Heller
Jul 19, 2003·Science·James C Carrington, Victor Ambros
Sep 17, 2003·Genes & Development·Eric MasséSusan Gottesman
Nov 19, 2003·Molecular Microbiology·Aixia ZhangSusan Gottesman
Apr 6, 2004·Current Opinion in Microbiology·Gisela StorzAixia Zhang
Oct 19, 2004·Annual Review of Microbiology·Susan Gottesman
Oct 23, 2004·Science·Eliot Marshall
Jun 15, 2005·Annual Review of Biochemistry·Gisela StorzKaren M Wassarman

❮ Previous
Next ❯

Citations

Jul 22, 2008·Nature Genetics·Natalie DutrowBradley R Cairns
Sep 21, 2010·Nature Methods·Shaomei HePhilip Hugenholtz
Nov 15, 2008·Nucleic Acids Research·Julia P SwierczMarie A Elliot
Dec 15, 2010·Nucleic Acids Research·Mark B SteadSidney R Kushner
Jan 27, 2012·Genes & Development·Peter C StirlingPhilip Hieter
Sep 4, 2012·Journal of Bacteriology·Mirjana MacvaninSankar Adhya
Aug 24, 2007·PLoS Biology·Erel LevineTerence Hwa
Apr 20, 2014·PLoS Genetics·Yujia A ChanPhilip Hieter
Feb 7, 2014·The Analyst·Blake N Johnson, Raj Mutharasan
Sep 13, 2007·Proceedings of the National Academy of Sciences of the United States of America·Lóránt SzékvölgyiGábor Szabó
Aug 17, 2010·Annual Review of Genetics·Maureen Kiley Thomason, Gisela Storz
Jul 12, 2014·Expert Review of Molecular Diagnostics·Zonglin HuChristopher A Elkins
Dec 25, 2015·Journal of Molecular Neuroscience : MN·James D MillsMichael Janitz
Jul 18, 2014·Nucleic Acids Research·Sébastien BrittonPatrick Calsou
Nov 18, 2015·Proceedings of the National Academy of Sciences of the United States of America·Francesca BersaniDaniel A Haber
Sep 18, 2009·Current Opinion in Microbiology·Cynthia Mira Sharma, Jörg Vogel
Jun 8, 2007·Current Opinion in Microbiology·Shoshy Altuvia
Oct 17, 2006·Molecular Microbiology·Sigrid C J De KeersmaeckerKathleen Marchal
Oct 3, 2013·The EMBO Journal·Verena PfeifferJoachim Lingner
Jun 21, 2013·Journal of Molecular Recognition : JMR·Damilola D PhillipsClinton E Leysath
Jan 8, 2011·Molecular Cell·Hannah E MischoNick J Proudfoot
Apr 19, 2015·BMC Research Notes·Zheng Z ZhangMichael R Lieber
Apr 5, 2014·Biochimica Et Biophysica Acta·André MaicherBrian Luke
Nov 8, 2014·Nucleic Acids Research·Zheng Z ZhangMichael R Lieber
Oct 17, 2007·FEMS Microbiology Letters·Tom CoenyeDavid W Ussery
Sep 19, 2014·PLoS Genetics·Matthias Groh, Natalia Gromak
Jul 22, 2016·PLoS Genetics·Julio C MoralesDavid A Boothman
Oct 19, 2017·The Journal of Cell Biology·Emily Yun-Chia ChangPeter C Stirling
Nov 1, 2017·Proceedings of the National Academy of Sciences of the United States of America·Zhong QianSankar Adhya
Oct 15, 2014·Journal of Bacteriology·Chen-Hsun TsaiLydia M Contreras
Sep 10, 2013·Nature Structural & Molecular Biology·Bettina BalkBrian Luke
Nov 27, 2014·The EMBO Journal·Arun K MankanVeit Hornung

❮ Previous
Next ❯

Methods Mentioned

BETA
co-immunoprecipitation
immunoprecipitations
PCR
in vitro transcription

Software Mentioned

Affymetrix GeneChip Operations Software GCOS
Affymetrix GeneChip ® Expression Analysis
GenePix Pro

Related Concepts

Related Feeds

Antibody Specificity

Antibodies produced by B cells are highly specific for antigen as a result of random gene recombination and somatic hypermutation and affinity maturation. As the main effector of the humoral immune system, antibodies can neutralize foreign cells. Find the latest research on antibody specificity here.