Microfluidic-based enzymatic on-chip labeling of miRNAs.
Small noncoding RNAs (sncRNAs) have moved from oddity to recognized important players in gene regulation. Next generation sequencing approaches discover more and more such molecules from a variety of different groups, but flexible tools translating this sequence information into affordable high-throughput assays are missing. Here we describe a microfluidic primer extension assay (MPEA) for the detection of sncRNAs on highly flexible microfluidic microarrays which combines several beneficial parameters: it can effortless incorporate any new sequence information; it is sensitive enough to work with as little as 20ng of total RNA and has a high level of specificity owing to a combination of a conventional hybridization assay and an enzymatic elongation step. Importantly, no labeling step is needed before hybridization and - because of its high sensitivity - no amplification is required. Both aspects ensure that no bias is introduced by such processes. Although the assay is exemplified with miRNAs, the flexibility of the technology platform allows the analysis of any type of sncRNA, such as piRNAs.
Vorwerk
Sonja
S
febit biomed GmbH, Im Neuenheimer Feld 519, 69120 Heidelberg, Germany. sonja.vorwerk@febit.de
Ganter
Kerstin
K
Cheng
Yang
Y
Hoheisel
Jörg
J
Stähler
Peer F
PF
Beier
Markus
M
D017931:DNA Primers / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012260:Ribonucleases / Q000235:genetics*; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods
D017931:DNA Primers; D035683:MicroRNAs; D012260:Ribonucleases