Abstract
In this study, we report the development of a dual extraction protocol for RNA and lipids, including phospholipids, endocannabinoids, and arachidonic acid, at high spatial resolution, e.g., brain punches obtained from whole frozen brains corresponding to four brain subregions: dorsal hippocampus, ventral hippocampus, basolateral amygdala, and hypothalamus. This extraction method combined with LC/multiple reaction monitoring for lipid quantifi-cation and quantitative PCR for RNA investigation allows lipidomic and transcriptomic profiling from submilligram amounts of tissue, thus benefiting the time and animal costs for analysis and the data reliability due to prevention of biological variability between animal batches and/or tissue heterogeneity, as compared with profiling in distinct animal batches. Moreover, the method allows a higher extraction efficiency and integrity preservation for RNA, while allowing concurrently quantitative analysis of low and high abundant lipids. The method was applied for brain punches obtained 1 h after kainic acid-induced epileptic seizures in mice (n = 10) compared with controls (n = 10), and enabled the provision of valuable new insights into the subregional lipid and RNA changes with epilepsy, highlighting its potential as a new viable tool in quantitative neurobiology.
Highlights
In this study, we report the development of a dual extraction protocol for RNA and lipids, including phospho lipids, endocannabinoids, and arachidonic acid, at high spa tial resolution, e.g., brain punches obtained from whole frozen brains corresponding to four brain subregions: dor sal hippocampus, ventral hippocampus, basolateral amyg dala, and hypothalamus
For quantitative multimolecular interrogations in brain punches, such as mRNAs and lipids or proteins and lipids, the sample amount of a brain punch [34] is not sufficient to efficiently extract two molecular classes. In such cases distinct batches of animals or different brain hemispheres are used, which is laborious, cost- and time-ineffective, and renders bias in molecular correlates due to variability between animal batches or brain hemispheres. To circumvent these limitations and expedite the targeted lipidomic and transcriptomic investigation in the brain at increased spatial resolution, we developed a method for simultaneous extraction of eCBs and arachidonic acid (AA), PLs, and RNA from a single tissue sample obtained by brain punching
Continuing our epilepsy research [8], we aimed at unraveling subregional lipid and mRNA fingerprints in brain punches of dorsal hippocampus, ventral hippocampus, basolateral amygdala (BLA), and hypothalamus (HYP) at acute epileptic seizure state
Summary
We report the development of a dual extraction protocol for RNA and lipids, including phospho lipids, endocannabinoids, and arachidonic acid, at high spa tial resolution, e.g., brain punches obtained from whole frozen brains corresponding to four brain subregions: dor sal hippocampus, ventral hippocampus, basolateral amyg dala, and hypothalamus. Interrogations in neurological diseases often require qualitative and quantitative molecular profiling at higher spatial resolution in the brain to more precisely localize the functional (sub)areas of the brain or cell populations involved in the disease This aspect has recently been emphasized to be of high relevance in epilepsy research [8]. In such cases distinct batches of animals or different brain hemispheres are used, which is laborious, cost- and time-ineffective, and renders bias in molecular correlates due to variability between animal batches or brain hemispheres To circumvent these limitations and expedite the targeted lipidomic and transcriptomic investigation in the brain at increased spatial resolution, we developed a method for simultaneous extraction of eCBs and arachidonic acid (AA) (collectively referred to as eCBs), PLs, and RNA from a single tissue sample obtained by brain punching. Combined with lipid MSI analysis, our study provides new insights into the subregional and/or cellular lipid dynamics caused by epileptic seizures
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