High-throughput confirmation of differential display PCR results using reverse Northern blotting.
Nylon filter arrays spotted with differential display PCR (DD-PCR) clones and hybridized with radiolabeled cRNA generated from the source RNA pool (reverse Northern blot) provide a high-throughput means to screen clones for artifacts. Reverse Northern blots also confirm differential gene expression in parallel and require modest quantities of the source RNA pool. We describe a strategy to screen multiple candidates from DD-PCR by high-throughput ligation and transformation, followed by reverse Northern blotting. Purification of re-amplified DD-PCR clones and fabrication of nylon arrays was facilitated by a batch-processing protocol using the widely available Biomek laboratory robot and Bioworks scripts (available from the authors). A strategy to screen out DD-PCR product artifacts of an inappropriate size was also employed. Using these approaches, we identified several mRNAs that are differentially expressed in response to venlafaxine, fluoxetine or desipramine antidepressant treatment in rat C6 glioma cell lines and are candidates for full length clone isolation using 5'-RACE. Such an approach provides a rapid means to eliminate the high percentage of false positive clones from DD-PCR and enables independent confirmation of differential gene expression patterns generated by various experimental conditions.
Dilks
Daniel W
DW
Wyeth Research, Neuroscience, CN-8000, Princeton, NJ 08543-8000, USA.
Ring
Robert H
RH
Khawaja
Xavier Z
XZ
Novak
Thomas J
TJ
Aston
Christopher
C
D000818:Animals; D000928:Antidepressive Agents / Q000494:pharmacology*; D016477:Artifacts; D015152:Blotting, Northern / Q000379:methods*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003626:Data Display; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D009457:Neuroglia / Q000187:drug effects; D050484:Norepinephrine Plasma Membrane Transport Proteins; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D011941:Receptors, Adrenergic / Q000096:biosynthesis / Q000235:genetics; D011985:Receptors, Serotonin / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D050486:Serotonin Plasma Membrane Transport Proteins; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D000928:Antidepressive Agents; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D012333:RNA, Messenger; D011941:Receptors, Adrenergic; D011985:Receptors, Serotonin; C493227:SLC6A2 protein, human; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493226:Slc6a2 protein, rat; C493232:Slc6a4 protein, rat; D027981:Symporters