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A microfluidic multiwell chip for enzyme-free detection of mRNA from few cells
- Haider, Michaela, Ji, Bozhi, Haselgrübler, Thomas, Sonnleitner, Alois, Aberger, Fritz, Hesse, Jan
- Biosensors & bioelectronics 2016 v.86 pp. 20-26
- nucleic acid hybridization, messenger RNA, gene overexpression, complementary DNA, pancreatic neoplasms, light microscopy, stem cells, reverse transcription, genes, gene expression regulation, biosensors
- Isogenic cell populations possess heterogeneous gene expression patterns. Most methods for mRNA expression analysis start with the reverse transcription of mRNA into cDNA, a process that can introduce strong signal variations not related to the actual mRNA levels. Miniaturized lab-on-a-chip systems offer properties – e.g. low sample dilution, low contamination – that enable new reaction schemes for molecular analyses. To enable transcription-free mRNA expression analysis of few single cells, a one-step cell lysis, target labelling and hybridisation approach as well as a corresponding passive multiwell chip with a volume of 25.5 nL/well were developed. The method enabled the parallel analysis of up to 96 samples and 6 target genes per sample. Preceding light microscopy of the living cells allowed correlating mRNA levels and cell number. As a proof-of-principle, the pancreatic cancer cell line Panc-1 was investigated for expression heterogeneity of a reference gene plus 5 genes reported to be overexpressed in cancer stem cells (CSCs). A good correlation (r(51)=0.739, p<0.001; rs(51)=0.744, p<0.001) between the cell number per well and the number of detected reference gene mRNA confirmed the proper function of the device. Moreover, a heterogeneous expression of the CSC-associated target genes was found which matched well with reports on the presence of CSCs in the Panc-1 cell line.