ac302128u_si_005.mp4 (10.17 MB)
Multiplexed Detection of mRNA Using Porosity-Tuned Hydrogel Microparticles
media
posted on 2012-11-06, 00:00 authored by Nak Won Choi, Jungwook Kim, Stephen C. Chapin, Thao Duong, Elaine Donohue, Pramod Pandey, Wendy Broom, W. Adam Hill, Patrick S. DoyleTranscriptional profiling, which is directly or indirectly associated
with expressed protein levels, has been used in various applications
including clinical prognosis and pharmaceutical investigation of drug
activities. Although the widely used reverse transcription polymerase
chain reaction (RT-PCR) allows for the quantification of absolute
amounts of mRNA (mRNA) from inputs as small as a single cell, it is
an indirect detection method that requires the amplification of cDNA
copies of target mRNAs. Here, we report the quantification of unmodified
full-length transcripts, using poly(ethylene) glycol diacrylate (PEGDA)
hydrogel microparticles synthesized via stop flow lithography (SFL).
We show that PEG600 serves as an effective porogen to allow for the
capture of large (∼1000–3700 nt long) mRNAs. Our relatively
simple hydrogel-based mRNA detection scheme uses a multibiotinylated
universal label probe and provides assay performance (limit of detection
of ∼6 amol of an in-vitro-transcribed model target) comparable
to an existing commercial bead-based technology that uses branched
DNA (bDNA) signal amplification. We also demonstrate a 3-plex mRNA
detection, without cross-reactivity, using shape-encoded “intraplex”
hydrogel microparticles. Our ability to tune the porosity of encoded
hydrogel microparticles expands the utility of this platform to now
quantify biomacromolecules ranging in size from large mRNAs to small
miRNAs.