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Expressional potency of mRNAs encoding receptors and voltage-activated channels in the postmortem rat brain
1Department of Psychobiology, University of California, Irvine 92717.
Abstract:
The stability and integrity of mRNAs encoding neurotransmitter receptors and voltage-activated channels in the postmortem rat brain was investigated by isolating poly(A)+ mRNA, injecting it into Xenopus oocytes, and then examining the expression of functional neurotransmitter receptors and voltage-activated channels in the oocyte membrane by electrophysiological recording. This approach was also used to assess the stability of mRNAs in brains that were incubated in oxygenated mammalian Ringer's solution for various lengths of time and from brains that were freshly frozen and then thawed at room temperature. Oocytes injected with mRNA from up to 21-hr postmortem brains gave large agonist- and voltage-activated responses, indicating that mRNAs encoding neurotransmitter receptors and voltage-activated channels are relatively stable in postmortem brain tissue. In contrast, oocytes injected with mRNA from brains incubated in Ringer's solution exhibited smaller responses, and oocytes injected with mRNA from tissue that was frozen and then thawed displayed very small or undetectable responses. Northern blot analysis using a nucleic acid probe for rat brain Na(+)-channel mRNA indicated that the size of the Na+ currents in injected oocytes reflected the levels of mRNA for Na+ channels in the different mRNA preparations. Thus, the expressional potency of mRNAs encoding neurotransmitter receptors and voltage-activated channels is quite stable in postmortem brains in situ, but it is reduced if the brains are kept in oxygenated saline, and freezing and thawing of tissue results in rapid degeneration of mRNA.
Insights
Messenger RNA (mRNA) for brain receptors and channels remains stable in postmortem rat brains for up to 21 hours. However, mRNA integrity rapidly declines when brains are stored in saline or subjected to freezing and thawing.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Postmortem tissue analysis is crucial for understanding brain function.
- The stability of mRNA in postmortem brain tissue is critical for accurate molecular studies.
- Neurotransmitter receptors and voltage-activated channels are key components of neuronal signaling.
Purpose of the Study:
- To investigate the stability and integrity of mRNAs encoding neurotransmitter receptors and voltage-activated channels in postmortem rat brains.
- To assess how different postmortem storage conditions affect mRNA integrity.
- To determine the functional expression of these mRNAs in Xenopus oocytes.
Main Methods:
- Isolation of poly(A)+ mRNA from postmortem rat brains.
- Injection of mRNA into Xenopus oocytes.
- Electrophysiological recording to assess functional receptor and channel expression.
- Northern blot analysis to confirm mRNA levels.
Main Results:
- Oocytes injected with mRNA from brains up to 21 hours postmortem showed robust functional responses.
- mRNA from brains incubated in Ringer's solution yielded diminished responses.
- mRNA from frozen and thawed brains resulted in very small or undetectable responses.
- Northern blot confirmed that Na+ channel mRNA levels correlated with observed Na+ currents.
Conclusions:
- mRNA encoding neurotransmitter receptors and voltage-activated channels is relatively stable in postmortem rat brains in situ.
- Storage in oxygenated saline reduces mRNA potency.
- Freezing and thawing leads to rapid mRNA degeneration.