ISH-guided freeze-matrix assisted punches: Technique for extracting punches from thin slide-mounted tissues for DNA methylation analysis
Molecular and Behavioral Neuroscience Institute, Department of Psychiatry, and Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI, USA.BioTechniques (Impact Factor: 2.95). 09/2012; 53(3):175-80. DOI: 10.2144/0000113912
Dissection of discrete brain regions for molecular analysis is complicated by trade-offs between accuracy, flexibility, and costs. We developed a flexible and cost-effective method, in situ hybridization (ISH) guided freeze-matrix assisted punches (IFAP), for extracting nanogram quantities of DNA from slide-mounted sections as thin as 12 μm. Using ISH to localize regions of interest, tissue is targeted by applying a small bead of M-1 embedding matrix onto cryosections, snap-freezing, and collecting the beads for nucleic acid purification. The method quantitatively recovers RNA and DNA usable for PCR and DNA methylation analysis.
- [Show abstract] [Hide abstract]
ABSTRACT: Adverse early life experiences (aELEs), such as child abuse, neglect, or trauma, increase lifetime vulnerability for mental illness. In this study, aELEs were modeled in c57bl/6 mice using the maternal separation (MS) paradigm, in which pups were separated for 180min/day (MS180), 15min/day (MS15), or left undisturbed (AFR) from postnatal day 2-14. As adults, pups that experienced MS15 or MS180 demonstrated decreases in tryptophan hydroxylase 2 (TPH2) and serotonin transporter mRNA in the dorsal raphe dorsalis and ventralis, and increases in glucocorticoid receptor mRNA in the dentate gyrus of the hippocampus. To investigate factors underlying shared expression between MS conditions, dam on-nest time and DNA methylation at the TPH2 promoter and 5' UTR were assessed. Post-reunion on-nest time increased as a function of separation duration, potentially serving as a mitigating factor underlying similar expression between MS conditions. TPH2 DNA methylation remained unchanged, suggesting changes in TPH2 mRNA are not mediated by changes in DNA methylation of this region. The shared pattern of expression between MS15 and MS180 conditions suggests a species- or strain- specific response to MS unique to c57bl/6 mice.Brain research 03/2013; 1515. DOI:10.1016/j.brainres.2013.03.032 · 2.84 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In situ hybridization-assisted punches (IFAP) are a low-cost method for extracting tissue from frozen slide-mounted sections as thin as 12 μm. The method synergizes well with standard histological workflows and uses in situ hybridization to target corresponding slide-mounted cryosections that contain the region of interest. Liquid beads of M-1 embedding matrix are applied and snap frozen, binding the matrix to the underlying tissue. Bead-tissue complexes are removed and DNA extracted using a high-salt method. IFAP-extracted DNA is suitable for downstream DNA methylation analysis.Methods in Molecular Biology 09/2014; 1211:237-243. DOI:10.1007/978-1-4939-1459-3_19 · 1.29 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.