Antisense RNA Amplification: A Linear Amplification Method for Analyzing the mRNA Population from Single Living Cells

Methods : a Companion to Methods in Enzymology
J Phillips, J Eberwine

Abstract

Cell-specific gene expression is a fundamental area of investigation of various disciplines, but is often complicated by cell heterogeneity, paucity of material, or limits of detection for low-abundance mRNAs. Antisense RNA (aRNA) amplification presents a linear amplification method for analyzing the mRNA population from single living cells. Patch clamp electrodes containing reverse transcriptase, dNTPS, and a poly(T) primer modified 5' with a T7 RNA polymerase promoter sequence are used to isolate the cytoplasmic contents of individual living cells. The cDNAs transcribed therefore contain the T7 promoter sequence. Following subsequent processing for second-strand cDNA synthesis, T7 polymerase is used for amplification, which results in a 2000-fold amplification of antisense RNA. A second round of amplification results in a 10(6)-fold amplification of the initial material. The aRNA amplification method may be used in conjunction with other techniques. Electrophysiology may be conducted first to examine functional properties. Reverse Northerns may be performed, using the aRNA as a probe to identify specific cDNAs loaded onto a slot blot. PCR may be conducted to determine splice variants. Differential display and library construc...Continue Reading

Citations

Apr 23, 2005·Nature Methods·Jennie E LarkinJohn Quackenbush
Jul 19, 2006·Nature Reviews. Genetics·Jian-Bing FanKevin L Gunderson
Dec 26, 2012·Proceedings of the National Academy of Sciences of the United States of America·Xinghua PanSherman M Weissman
Sep 5, 2007·Assay and Drug Development Technologies·Mingjie ZhouThomas L Fare
Apr 19, 2012·Briefings in Functional Genomics·Abdullah A Y Gibriel
Feb 5, 2005·Bioinformatics·Marie-Laure Martin-MagnietteJean-Jacques Daudin
Aug 24, 2012·Journal of the Royal Society, Interface·James EberwineJunhyong Kim
Feb 1, 2002·Biochemical and Biophysical Research Communications·Yosuke MizunoYasushi Okazaki
Sep 28, 2005·Proceedings of the National Academy of Sciences of the United States of America·N DevidzeD W Pfaff
Mar 7, 2002·Proceedings of the National Academy of Sciences of the United States of America·Klára KitajkaTibor Farkas
Nov 20, 2002·Proceedings of the National Academy of Sciences of the United States of America·Chang-Zheng ChenHarvey F Lodish
Jun 12, 2003·Journal of Neuroimmunology·Fumiko KoikeTakashi Yamamura
Nov 25, 2004·Progress in Biophysics and Molecular Biology·Simon HughesJeremy Squire
Oct 3, 2003·Biochemical and Biophysical Research Communications·Yasuhiro TomaruYoshihide Hayashizaki
Jan 31, 2004·Biochemical and Biophysical Research Communications·Takashi ShimojiKenichi Shinomiya
Jan 28, 2004·Comptes rendus biologies·Jiangning LiRoger E Bumgarner
Apr 6, 2004·Progress in Neurobiology·David HinkleJames Eberwine
Nov 21, 2007·Expert Review of Molecular Diagnostics·Johanna R Mora, Robert C Getts
May 19, 2006·Expert Review of Molecular Diagnostics·Clelia PeanoCristina Battaglia
Nov 26, 2015·Fertility and Sterility·Eleni MantikouMark de Jong
Aug 21, 2010·Analytical Biochemistry·D NawarathnaH Kumar Wickramasinghe
May 18, 2010·Journal of Virological Methods·Antonio TiberiniAhmed Hadidi
Nov 17, 2007·Journal of Plant Physiology·Elke LieckfeldtJoachim Fisahn
Jul 19, 2015·Nucleic Acids Research·Justus M Kebschull, Anthony M Zador
Sep 29, 2005·Molecular Nutrition & Food Research·Bettina Spielbauer, Frank Stahl
Aug 19, 2004·The Plant Journal : for Cell and Molecular Biology·Rob AlbaJim Giovannoni
Mar 26, 2005·Molecular Reproduction and Development·So Hyun LeeChristopher K Tuggle
Oct 24, 2006·Epilepsia·Peter B Crino, Albert J Becker
Dec 22, 2006·The Plant Journal : for Cell and Molecular Biology·Masanao SatoFumiaki Katagiri
Oct 7, 2004·Analytical Biochemistry·Pamela R MollKlaus Richter
Jul 28, 2006·Analytical Biochemistry·Michelle Alvarez, Jack Ballantyne
Nov 1, 2005·Ageing Research Reviews·Theresa Joseph Kannanayakal, James Eberwine
Aug 27, 2005·Clinica Chimica Acta; International Journal of Clinical Chemistry·David ZhangBingjiao Yin
Jul 11, 2002·Biochemical Pharmacology·Françoise de LonguevilleJosé Remacle
Nov 24, 2004·Biochemical and Biophysical Research Communications·Jik Young ParkSuk Woo Nam
Feb 9, 2007·Differentiation; Research in Biological Diversity·Vasker BhattacherjeeM Michele Pisano

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