| dc.description.abstract | Receptor subunit composition can be examined at the mRNA (gene expression) level. Profiling gene expression in acutely isolated neurons from adult animals offers a potentially powerful approach to determining GABAₐ receptor subunit composition and sensitivity. Recently antisense RNA (aRNA) has been amplified from total cellular mRNA present in single cells (2). First, single live neurons are isolated from the adult rat brains. High-resistance (giga Ohm) patch-clamp recordings of whole cell currents may be obtained from the cells analyzed for mRNA expression. Following electrophysiology, the cell is aspirated into the patch pipette which contains avian myeloblastoma virus reverse transcriptase, nucleotides, oligo (dT)-T7 promoter containing primer, and buffer for cDNA synthesis. The resulting cDNA's are then processed to double stranded blunt ended molecules; at this stage the cDNA population should represent the relative quantity of distinct mRNA species present in the cell at the time of aspiration. Linear amplification is accomplished using T7 RNA polymerase to produce single stranded antisense RNA. A second round of amplification can yield a million fold increase of the original message (2). Reverse Northern hybridizations are then used to assay the amplification product for expression of specific genes.
The polymerase chain reaction is becoming an increasingly common method for expression analysis in single cells (6). Amplification of antisense RNA, however, offers advantages over PCR. Oligonucleotide primers used in PCR often prime DNA synthesis with differing efficiency (8). Differential primer efficiency is then exponentially amplified throughout thermal cycling. Accordingly, reactions in which multiple genes are assayed are difficult to quantify. Furthermore, total primer sets in one reaction rarely exceeds three, limiting the number of genes which can be assayed in a single cell. Antisense RNA alleviates these difficulties by linearly amplifying total cellular mRNA. This yields a large product from which expression of multiple genes may be quantitated. | en |
