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

Analytical Biochemistry
J ThompsonD Gillespie

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 x 10(6) cells in concentrated cell lysates (10(8) cell...Continue Reading

References

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Citations

Jan 1, 1994·Journal of Medical Virology·L J van DoornW Quint
Dec 17, 1990·Clinica Chimica Acta; International Journal of Clinical Chemistry·E P Diamandis
Feb 1, 1992·Parasitology Today·S Dissanayake, W F Piessens
Sep 1, 1990·Veterinary Microbiology·D Gillespie
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Oct 22, 2005·Preparative Biochemistry & Biotechnology·Charoula Psallida, Dimitris Argyropoulos
Jul 19, 2011·Journal of Biomedicine & Biotechnology·Marie J Archer, Baochuan Lin
Oct 1, 1990·Molecular and Cellular Probes·T JalavaM Ranki
Jan 1, 1989·Journal of Clinical Laboratory Analysis·R SolomonD Gillespie
Aug 1, 1990·Clinical Biochemistry·J D Thompson, D Gillespie

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