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A noise-free molecular hybridization procedure for measuring RNA in cell lysates
Journal article   Peer reviewed

A noise-free molecular hybridization procedure for measuring RNA in cell lysates

James Thompson, Robert Solomon, Michael Pellegrino, Koji Sakai, Mark Lewin, Maggie Feild, Margaret Castrovinci, Lawrence Sacramone, David Gillespie and Jervis Walter Thompson
Analytical biochemistry, v 181(2), pp 371-378
1989
DOI: https://doi.org/10.1016/0003-2697(89)90257-1
PMID: 2479299
Featured in Collection :   UN Sustainable Development Goals @ Drexel

Abstract

A solution hybridization technique was designed to measure RNA abundance in crude cell lysates and at the same time to maximize confidence that signals resulted from true molecular hybridization. Cell lysates were prepared in 5 m guanidine thiocyanate, then RNA molecules in the lysates were hybridized with two probes, a 32P-labeled RNA “label probe” which provided signal and an oligodeoxyribonucleotide “capture probe” containing a poly(dA) tail which provided a mechanism for selective purification. Ternary hybrids were “captured” on oligo(dT)-coated superparamagnetic beads through a readily reversible interaction with the poly(dA) of the capture probe. RNA did not bind to dT beads through poly(A) under the capture conditions used. Hybrids were purified through cycles of capture on and release from dT beads, with each cycle yielding a 100- to 1000-fold reduction in noise (unhybridized label probe) and a 50–90% recovery of signal (hybridized label probe). Noise was driven below detectable limits after three cycles of capture, thereby improving the sensitivity of measuring target RNA. As few as 15,000 target molecules, 15 fg of a 3-kb RNA, was detectable in the equivalent of 2 × 10 6 cells in concentrated cell lysates (10 8 cells/ml). Since hybridization with both probes was required in order to yield a signal, hybridization specificity could be adjusted with either or both probes. The greater specificity and lack of noise increased confidence that the signal was proportional to the amount of RNA of interest.

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20 citations in Web of Science
21 citations in Scopus

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Collaboration types
Domestic collaboration
Web of Science research areas
Biochemical Research Methods
Biochemistry & Molecular Biology
Chemistry, Analytical
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