The hippocampus is a complex structure that is more sensitive to epileptogenic stimuli than other regions in the brain. When the hippocampal cells become epileptic, they show altered electrical activity and disturbed Ca2+ homeostasis over the short and long term(1)
. Because the plasma membrane Ca2+ ATPase (PMCA) efficiently extrudes Ca2+ from eukaryotic cells(2)
, we examined the expression of PMCA transcripts in neurons derived from the hippocampus. Since the level of PMCA is strictly regulated in excitable cells, the more than 20 enzyme isoforms produced by alternative splicing of four genes at sites A and C become important (Figure 1; (2)
). Single-cell RT-PCR is a sensitive technique that can detect transcripts, but it involves risks such as contamination or the need to perform nested PCR. Here, through an interdisciplinary approach involving electrophysiological and molecular biology techniques(4)
, we took advantage of the highly sensitive AccessQuick™ RT-PCR System (Cat.# A1703) to detect zeptomolar levels of PMCA transcripts. We analyzed PMCA transcripts generally considered to have poor expression levels in single hippocampal cells from the CA1 zone and used GAPDH as a housekeeping gene.
Materials and Methods
Male Wistar rats 20–25 days old were used, and brain slices were obtained in an appropriate artificial cerebrospinal fluid. Single hippocampal cells were visualized by infrared differential interference contrast (DIC) video microscopy and harvested using the whole-cell configuration, which permits us to document individual electrophysiological recordings and isolate a single cell by only applying negative pressure to the microelectrode (Figure 2). Normal hippocampal cells were collected in the presence of RNasin® Plus RNase Inhibitor (Cat.# N2611), frozen in liquid nitrogen and kept at –70ºC until further use.
Figure 2. Single cell from the CA1 area of the hippocampus visualized under a DIC infrared microscope using a whole-cell configuration.
One-step RT-PCRs were carried out in a final volume of 50µl following the AccessQuick™ RT-PCR System Product Information in an RNase-free environment. Each reaction contained 0.1µM of primers for each of the PMCA transcripts amplified. Primers used for site A were previously reported(5)
and we designed site C-specific primers using MacVector 6.5.3 software with rat sequences for the PMCA isoforms (PMCA1: NM_053311; PMCA2: NM_012508; PMCA3: NM_133288; PMCA4: NM_001005871).
For reverse transcription, samples were incubated at 45ºC for 45 minutes. PCR conditions were as follows: 94ºC for 2 minutes, followed by 45 cycles at 94ºC for 15 seconds, 55ºC for 30 seconds and 72ºC for 1 minute. PCR was concluded with a final extension step at 72ºC for 5 minutes, and the amplified samples were chilled at 4ºC. Twenty-five microliters of each reaction was loaded on a 4% agarose gel, separated by electrophoresis and visualized by ethidium bromide staining. For better sensitivity, the agarose gels were scanned in a Typhoon® 9400 scanner (GE Healthcare Bio-sciences) using fluorescence detection mode. The amplified fragments were sequenced to confirm their identity.
Employing single hippocampal cells, our data clearly show the presence of several PMCA transcripts edited at site A encoded by genes 1 (PMCA1A), 2 (PMCA2A), and 3 (PMCA3A) or at site C (PMCA1C; Figure 3, Panel B). The presence of nonspecific amplicons in the PMCA3A reaction suggests more stringent PCR conditions are needed.
This study provides a new application for the AccessQuick™ RT-PCR System that requires only one step to overcome the potential problems involved in single-cell RT-PCR techniques like contamination or performing nested PCR. Using this system, it is possible to analyze products from single cells on ethidium bromide-stained agarose gels.
This work was supported by CONACyT (Grant 47333/A-1) and DGAPA-UNAM (Grant IN228607/20) awarded to J. M-O.