Multiplexed screening assay for mRNA combining nuclease protection with luminescent array detection.
The principles and performance are described for the ArrayPlate mRNA assay, a multiplexed mRNA assay for high-throughput and high-content screening and drug development. THP-1 monocytes grown and subjected to compound treatments in 96-well plates were subjected to a multiplexed nuclease protection assay in situ. The nuclease protection assay destroyed all cell-derived mRNA, but left intact stoichiometric amounts of 16 target-specific oligonucleotide probes. Upon transfer of processed cell lysates to a microplate that contained a 16-element oligonucleotide array at the bottom of each well, the various probe species were separated by immobilization at predefined elements of the array. Quantitative detection of array-bound probes was by enzyme-mediated chemiluminescence. A high-resolution charge-coupled device imager was used for the simultaneous readout of all 1536 array elements in a 96-well plate. For the measurement of 16 genes in samples of 25000 cells, the average standard deviation from well to well within a plate was 8.6% of signal intensity and was 10.8% from plate to plate. Assay response was linear and reproducibility was constant for all detected genes in samples ranging from 1000 to 50000 cells. When THP-1 monocytes were differentiated with phorbol ester and subsequently activated with bacterial lipopolysaccharide that contained different concentrations of dexamethasone, dose-dependent effects of dexamethasone on the mRNA levels of several genes were observed.
Martel
Ralph R
RR
High Throughput Genomics, Inc, Tucson, AZ 85712, USA. rmartel@htgenomics.com
Botros
Ihab W
IW
Rounseville
Matthew P
MP
Hinton
James P
JP
Staples
Robin R
RR
Morales
David A
DA
Farmer
John B
JB
Seligmann
Bruce E
BE
D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007202:Indicators and Reagents; D008070:Lipopolysaccharides / Q000494:pharmacology; D008163:Luminescent Measurements; D009000:Monocytes / Q000187:drug effects; D023282:Nuclease Protection Assays / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000138:chemical synthesis; D010703:Phorbol Esters / Q000494:pharmacology; D012313:RNA; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D015203:Reproducibility of Results
D000893:Anti-Inflammatory Agents; D007202:Indicators and Reagents; D008070:Lipopolysaccharides; D009841:Oligonucleotides; D010703:Phorbol Esters; C034477:RNA primers; D012333:RNA, Messenger; D012313:RNA; D003907:Dexamethasone