A microfluidic multiwell chip for enzyme-free detection of mRNA from few cells.
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.
Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria.
Michaela
M
Haider
Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria.
Bozhi
B
Ji
Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria.
Thomas
T
Haselgrübler
Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria.
Alois
A
Sonnleitner
Cancer Cluster Salzburg, Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.
Fritz
F
Aberger
Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria. Electronic address: jan.hesse@cbl.at.
Jan
J
Hesse
D014408:Biomarkers, Tumor / Q000378:metabolism*; D045744:Cell Line, Tumor; D004798:Enzymes; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation; D056656:Lab-On-A-Chip Devices; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D046888:Tissue Array Analysis / Q000295:instrumentation*
D014408:Biomarkers, Tumor; D004798:Enzymes; D012333:RNA, Messenger
Cellular heterogeneity; Lab-on-a-chip; Microarray; Ultra-sensitive detection; mRNA expression