Solid phase gene extraction isolates mRNA at high spatial and temporal resolution.
Rapid, localized changes in gene expression require mRNA extraction at high temporal and spatial resolution. Current small-scale mRNA extractions depend on the removal of the cells/tissue from an organism or preserved specimens. What these methods have in common is that they are destructive and do not distinguish between genomic DNA and RNA. Therefore, extracted (m)RNA is typically contaminated by extracted cytoplasm, nuclear DNA, or other compounds, and the required purification leads to loss of especially low-abundant mRNA. The need to repeatedly remove mRNA from living material has led to the development of solid phase gene extraction (SPGE). SPGE sampling can be achieved using gene-specific or generic sequences and is not species-specific. Here we demonstrate the versatility and validity of this novel RNA extraction by simultaneously profiling nanos and bicoid mRNA in individual Drosophila eggs. The SPGE technique detects previously described distribution profiles of nanos and bicoid. Its low impact is underscored by the normal development of repeatedly sampled eggs. In our study, quantification of actin mRNA in germinating flax seeds linked gene expression to distinct developmental processes. These data demonstrate the universality of SPGE as a simple generic, analytical, and diagnostic procedure.
Scherp
Peter
P
Biology Department, University of Louisiana at Lafayette, Lafayette, LA 70504-2451, USA.
Hasenstein
Karl H
KH
D000199:Actins / Q000235:genetics; D000818:Animals; D004330:Drosophila / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics; D019597:Flax / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics; D010063:Ovum / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification*; D016601:RNA-Binding Proteins / Q000235:genetics; D052616:Solid Phase Extraction / Q000379:methods*; D015534:Trans-Activators / Q000235:genetics
D000199:Actins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D015534:Trans-Activators; C056478:bcd protein, Drosophila; C070574:nos protein, Drosophila