title: Multiplexed detection of mRNA using porosity-tuned hydrogel microparticles.
abstract: Transcriptional 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.
authors.affiliation: Novartis Institutes for Biomedical Research (NIBR), Cambridge, Massachusetts 02139, United States.
authors.forename: Nak Won
authors.initials: NW
authors.lastname: Choi
authors.forename: Jungwook
authors.initials: J
authors.lastname: Kim
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Chapin
authors.forename: Thao
authors.initials: T
authors.lastname: Duong
authors.forename: Elaine
authors.initials: E
authors.lastname: Donohue
authors.forename: Pramod
authors.initials: P
authors.lastname: Pandey
authors.forename: Wendy
authors.initials: W
authors.lastname: Broom
authors.forename: W Adam
authors.initials: WA
authors.lastname: Hill
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Doyle
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D019921:Biotinylation; D004247:DNA / Q000737:chemistry; D020100:Hydrogels / Q000737:chemistry*; D008863:Microspheres; D021141:Nucleic Acid Amplification Techniques; D011092:Polyethylene Glycols / Q000737:chemistry; D016062:Porosity; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D020100:Hydrogels; D012333:RNA, Messenger; D011092:Polyethylene Glycols; D004247:DNA