A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray

Abstract

A double-hybridization approach was developed for the enzyme-free detection of specific mRNA of a housekeeping gene. Targeted mRNA was immobilized by hybridization to complementary DNA capture probes spotted onto a microarray. A second hybridization step of Cy5-conjugated label DNA to another section of the mRNA enabled specific labeling of the target. Thus, enzymatic artifacts could be avoided by omitting transcription and amplification steps. This manuscript describes the development of capture probe molecules used in the transcription- and amplification-free analysis of RPLP0 mRNA in isolated total RNA. An increase in specific signal was found with increasing length of the target-specific section of capture probes. Unspecific signal comprising spot autofluorescence and unspecific label binding did not correlate with the capture length. An additional spacer between the specific part of the capture probe and the substrate attachment site increased the signal significantly only on a short capture probe of approximately 30 nt length.

RNA Biomarkers: Diagnostic and Prognostic Potentials and Recent Developments of Electrochemical Biosensors

“…These methods typically require some forms of amplification steps, cumbersome sample pretreatment procedure, and expensive instrumentation. Additionally, they often cannot amplify RNA sequences without a poly(A) tail due to the use of oligo(dT) primers for the amplification purpose . Additionally, relatively large sample volumes are required for sensing RNA biomarkers in body fluids to avoid sample heterogeneity, resulting from distinct physiological and systemic differences of clinical samples .…”

The Future of Biomarkers in Veterinary Medicine: Emerging Approaches and Associated Challenges

“…Special precautions must be taken when working with RNA to address the high risk of contamination such as the ubiquitous presence of RNases [ 75 ]. Furthermore, because oligo(dT) primers are used for amplification, they frequently cannot amplify RNA sequences without a poly(A) tail [ 76 ]. Moreover, to prevent sample heterogeneity caused by physiological and systemic changes in clinical samples, very high sample volumes are required for sensing RNA biomarkers in body fluids [ 77 ].…”

“…Electrochemical approaches, for example, have demonstrated ultra-high sensitivity and selectivity, as well as a high potential for multiplexed analysis in a point-of-care platform [ 81 , 82 ]. However, the functioning of electrochemical RNA sensors is still limited to proof-of-concept research, and various obstacles must be overcome before these technologies may be used in typical clinical settings [ 54 , 76 ].…”

Biosensor for the Characterization of Gene Expression in Cells

“…Gel electrophoresis and fluorescence intensity are used as external readouts, which contribute to the high cost and complexity of this method. Due to these limitations, the nucleic acid detection methods are non-suitable for point of care applications [17].…”