Effect of unlabeled helper probes on detection of an RNA target by bead-based sandwich hybridization

Abstract

Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site of the 5' end attached capture probe were found much more effective than helper probes targeting positions adjacent to the detection probe binding site. The difference is believed to be caused by a disruption of the RNA secondary structure in the area where the capture probe binds, thereby reducing structural interference from the bead. The use of additional helpers showed an additive effect. Using helpers at both sides of the capture and detection probes showed a 15- to 40-fold increase in hybridization efficiency depending on the target, thereby increasing the sensitivity of the hybridization assays. Using an electrical chip linked to the detection probe for the detection of p-aminophenol, which is produced by alkaline phosphatase, a detection limit of 2 x 10(-13) M mRNA molecules was reached without the use of a nucleic acid amplification step.

Specific magnetic isolation for direct detection of HPV16

“…As demonstrated here and reported elsewhere [27,28], the use of multiple capture probes enhances significantly the specific isolation. When compared with the non-mixed approach, overall higher purification efficiencies were observed for the mixed approach.…”

Transcriptional response of P. pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction

“…One of those is a magnetic bead-based sandwich hybridisation system, based on two specific oligonucleotide probes [3]. This system is advantageous over the conventional methods, Northern blot or slot blot, due to the high specificity and sensitivity, which are eventually improved by the use of unlabelled helper probes [4]. Furthermore, this system can be automated for high throughput analysis, and is also applicable with minor modifications for the analysis of antigens which is a major advantage in comparison to other techniques, such as Real Time RT-PCR.…”

Sandwich Hybridization Assay for Sensitive Detection of Dynamic Changes in mRNA Transcript Levels in Crude Escherichia coli Cell Extracts in Response to Copper Ions

“…This higher degree of sensitivity is probably owed to the helpers' assistance in opening up the secondary structure around the target where the probe is to bind. Additionally, there is an effect of base stacking between two adjacent terminal nucleotides from two oligonucleotides hybridizing next to each other (2). The shift away from beadbased sandwich hybridization toward SBSH has resulted in greater sensitivity of the assay and has facilitated liquid handling (17,19).…”

“…The use of helper probes alongside capture and detection probes increases the hybridization efficiency in sandwich hybridization by 15-to 40-fold as demonstrated previously (2). Detection probes were generated by 3Ј end labeling of oligonucleotides by using a second-generation DIG oligonucleotide 3Ј-end-labeling kit according to the instructions of the manufacturer (Roche, Mannheim, Germany).…”