Antisense RNA Amplification: A Linear Amplification Method for Analyzing the mRNA Population from Single Living Cells
Cell-specific gene expression is a fundamental area of investigation of various disciplines, but is often complicated by cell heterogeneity, paucity of material, or limits of detection for low-abundance mRNAs. Antisense RNA (aRNA) amplification presents a linear amplification method for analyzing the mRNA population from single living cells. Patch clamp electrodes containing reverse transcriptase, dNTPS, and a poly(T) primer modified 5' with a T7 RNA polymerase promoter sequence are used to isolate the cytoplasmic contents of individual living cells. The cDNAs transcribed therefore contain the T7 promoter sequence. Following subsequent processing for second-strand cDNA synthesis, T7 polymerase is used for amplification, which results in a 2000-fold amplification of antisense RNA. A second round of amplification results in a 10(6)-fold amplification of the initial material. The aRNA amplification method may be used in conjunction with other techniques. Electrophysiology may be conducted first to examine functional properties. Reverse Northerns may be performed, using the aRNA as a probe to identify specific cDNAs loaded onto a slot blot. PCR may be conducted to determine splice variants. Differential display and library construction may be employed to identify unknown or novel genes. Thus, the aRNA amplification may prove a valuable method for providing information on cell-specific gene expression in a variety of studies.
Phillips
J
Department of Pharmacology, University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, Pennsylvania, 19104-6084
Eberwine
JH