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Detecting PMCA Transcripts in Single Hippocampal Neurons

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RT-PCR should be performed in the absence as well as presence of the reverse transcriptase, to assess DNA contamination in the template RNA. In addition, "no-template" negative control reactions should always be performed.

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Abstract

RNA from single rat hippocampal neurons was amplified using the AccessQuick™ RT-PCR System to examine transcript expression of plasma membrane Ca2+ ATPase splice variants. The sensitivity of single-cell RT-PCR allowed detection of low-level transcripts using ethidium bromide-stained agarose gels.

Blanca Delgado-Coello1, Jorge Bravo-Martínez1,2 and Jaime Mas-Oliva1

1Department of Biochemistry, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México. México, D.F., México 2Department of Physiology, Facultad de Medicina, Universidad Nacional Autónoma de México. México, D.F., México
Publication Date: 2009

Introduction

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) (3) , 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.

Exon structure corresponding to alternative splicing options at sites A and C for the four PMCA genesFigure 1. Exon structure corresponding to alternative splicing options at sites A and C for the four PMCA genes.

Site A is near the amino terminus of PMCA, close to a phospholipid-sensitive region, and the site C sequence is near the domain containing the calmodulin-binding site toward the carboxy end of PMCA. Site C also has greater variability due to the presence of internal splicing donor sites in several exons. Alternatively spliced exons are indicated by blue boxes while those constitutively expressed are represented by white boxes.

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.

Single cell from the CA1 area of the hippocampus visualized under a DIC infrared microscope using a whole-cell configuration. tpub010_fig2Figure 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) (6) 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.

Results

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.

Representative agarose gels showing transcripts amplified with the AccessQuick RT-PCR SystemFigure 3. Representative agarose gels showing transcripts amplified with the AccessQuick™ RT-PCR System.

Panel A. GAPDH transcripts (125bp) amplified from two hippocampal cells (lane 1) or only one cell (lane 2). Lane M, Low DNA Mass Ladder (Invitrogen). Panel B. PMCA transcripts expressed in independent single hippocampal cells. Lane M, Low DNA Mass Ladder (Invitrogen); lane 1, 120bp PMCA1A fragment; lane 2, 300bp PMCA2A fragment; lane 3, 300bp PMCA3A fragment; lane 4, 66bp PMCA1C fragment; lane 5, negative control omitting reverse transcriptase.

Conclusion

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.

Acknowledgement

This work was supported by CONACyT (Grant 47333/A-1) and DGAPA-UNAM (Grant IN228607/20) awarded to J. M-O.

References

  1. DeLorenzo, R.J., Sun, D.A. and Deshpande, L.S. (2005) Cellular mechanisms underlying acquired epilepsy: The calcium hypothesis of the induction and maintenance of epilepsy. Pharmacol. Ther. 105, 229–66.
  2. Strehler, E.E. et al. (2007) Plasma membrane Ca2+ ATPases as dynamic regulators of cellular calcium handling. Ann. N.Y. Acad. Sci. 1099, 226–36.
  3. Delgado-Coello, B., Trejo, R. and Mas-Oliva, J. (2006) Is there a specific role for the plasma membrane Ca2+-ATPase in the hepatocyte? Mol. Cell. Biochem. 285, 1–15.
  4. Sucher, N.J. et al. (2000) Genes and channels: Patch/voltage-clamp analysis and single-cell RT-PCR. Cell Tissue Res. 302, 295–30.
  5. Mamic, T.M. et al. (2000) PMCA1 mRNA expression in rat aortic myocytes: A real time RT-PCR study. Biochem. Biophys. Res. Comm. 276, 1024–7.
  6. Souza, K.L. et al. (2004) Cytokines activate genes of the endocytotic pathway in insulin-producing RINm5F cells. Diabetologia 47, 1292–302.

How to Cite This Article

Delgado-Coello, B., Bravo-Martínez, J. and Mas-Oliva, J. Detecting PMCA Transcripts in Single Hippocampal Neurons. [Internet] 2009. [cited: year, month, date]. Available from: http://www.promega.com/resources/pubhub/detecting-pmca-transcripts-in-single-hippocampal-neurons/

Delgado-Coello, B., Bravo-Martínez, J. and Mas-Oliva, J. Detecting PMCA Transcripts in Single Hippocampal Neurons. Promega Corporation Web site. http://www.promega.com/resources/pubhub/detecting-pmca-transcripts-in-single-hippocampal-neurons/ Updated 2009. Accessed Month Day, Year.

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Figures

Exon structure corresponding to alternative splicing options at sites A and C for the four PMCA genesFigure 1. Exon structure corresponding to alternative splicing options at sites A and C for the four PMCA genes.

Site A is near the amino terminus of PMCA, close to a phospholipid-sensitive region, and the site C sequence is near the domain containing the calmodulin-binding site toward the carboxy end of PMCA. Site C also has greater variability due to the presence of internal splicing donor sites in several exons. Alternatively spliced exons are indicated by blue boxes while those constitutively expressed are represented by white boxes.

Single cell from the CA1 area of the hippocampus visualized under a DIC infrared microscope using a whole-cell configuration. tpub010_fig2Figure 2. Single cell from the CA1 area of the hippocampus visualized under a DIC infrared microscope using a whole-cell configuration.
Representative agarose gels showing transcripts amplified with the AccessQuick RT-PCR SystemFigure 3. Representative agarose gels showing transcripts amplified with the AccessQuick™ RT-PCR System.

Panel A. GAPDH transcripts (125bp) amplified from two hippocampal cells (lane 1) or only one cell (lane 2). Lane M, Low DNA Mass Ladder (Invitrogen). Panel B. PMCA transcripts expressed in independent single hippocampal cells. Lane M, Low DNA Mass Ladder (Invitrogen); lane 1, 120bp PMCA1A fragment; lane 2, 300bp PMCA2A fragment; lane 3, 300bp PMCA3A fragment; lane 4, 66bp PMCA1C fragment; lane 5, negative control omitting reverse transcriptase.