Brain banks: benefits, limitations and cautions concerning the use of post-mortem brain tissue for molecular studies

Abstract

Brain banks are facilities providing an interface between generous donation of nervous tissues and research laboratories devoted to increase our understanding of the diseases of the nervous system, discover new diagnostic targets, and develop new strategies. Considering this crucial role, it is important to learn about the suitabilities, limitations and proper handling of individual brain samples for particular studies. Several factors may interfere with preservation of DNA, RNA, proteins and lipids, and, therefore, special care must be taken first to detect sub-optimally preserved tissues and second to provide adequate material for each specific purpose. Basic aspects related with DNA, RNA and protein preservation include agonal state, post-mortem delay, temperature of storage and procedures of tissue preservation. Examination of DNA and RNA preservation is best done by using bioanalyzer technologies instead of less sensitive methods such as agarose gels. Adequate RNA preservation is mandatory in RNA microarray studies and adequate controls are necessary for proper PCR validation. Like for RNA, the preservation of proteins is not homogeneous since some molecules are more vulnerable than others. This aspect is crucial in the study of proteins including expression levels and possible post-translational modifications. Similarly, the reliability of functional and enzymatic studies in human post-mortem brain largely depends on protein preservation. Much less is known about other aspects, such as the effects of putative deleterious factors on epigenetic events such as methylation of CpGs in gene promoters, nucleosome preservation, histone modifications, and conservation of microRNA species. Most brains are appropriate for morphological approaches but not all brains are useful for certain biochemical and molecular studies.

Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the “adenosine hypothesis”

“…However, special care must be taken as there are several technical limitations for molecular studies, such as time after death and storage temperature [42]. For these reasons, we aimed to assess the effect of postmortem delay on ecto-nucleotidase activity.…”

Translational Research in Pediatrics IV: Solid Tissue Collection and Processing

“…225 Regardless of the tissue origin (either biopsy/surgical or postmortem), snap-freezing with storage at -80°C or below are ideal to maintain the integrity of biochemical molecules. 207,210,227 Tissue samples are often fixed in formaldehyde or formalin before being embedded in paraffin wax for sectioning and subsequent histologic examination. Fixed tissue is most commonly used for protein localization and morphology studies.…”

“…For example, brain RNA and protein degradation associated with postmortem delay is dependent on the storage temperature. 210,234,268 ADORA2B, adenosine A2B receptor; CAPN2, calpain-2 catalytic subunit 2; CAR, androstane receptor; CCL, chemokine (C-C motif) ligand; CD3, cluster of differentiation 3; CD4, cluster of differentiation 4; CD20, cluster of differentiation 20; CD68, cluster of differentiation 68; CoQ10, coenzyme Q10; CpG, C-phosphate-G (cytosine nucleotide sites); 13 C-UBT, 13 C-urea breath test; c-Src, Proto-oncogene tyrosine-protein kinase Src; CTCF, CCCTC-binding factor; CXCL1, the chemokine (C-X-C motif) ligand 1; DCX, doublecortin; EGFR, epidermal growth factor receptor; EPHX2, epoxide hydrolase 2; FACS, fl uorescence-activated cell sorting; FAK, focal adhesion kinase; FISH, fl uorescence in situ hybridization; fqPCR, fl uorescent quantitative PCR; Gli-RFP reporter, Gli luciferase reporter; GM-CSF, granulocyte-macrophage colony-stimulating factor; GR, glucocorticoid receptor; ICC, immunocytochemistry; IF, immunofl uorescence; IHC, immunohistochemistry; IL, interleukin; iNOS, inducible nitric oxide synthase; HA117, a novel multidrug resistance gene; H&E, hematoxylin and eosin; Hh, Hedgehog; HHV-6, human herpesvirus 6; HIV-1, HIV type 1; HNF1α, hepatocyte nuclear factor 1α; HNF4α, hepatocyte nuclear factor 4α; HPLC, high-performance liquid chromatography; iPSCs, induced pluripotent stem cells; Ki67, antigen identifi ed by monoclonal antibody; MDR1, multidrug resistance 1; MeDIP, methylated DNA immunoprecipitation; MEHP, mono-2-ethylhexylphthalate; MLCK, myosin light-chain kinase; MRP2, multidrug resistance protein 2; NANOG, homeobox protein NANOG; non-POPs, non-persistent organic pollutants; 4-NP, 4-nonylphenol; OATP1B1, organic anion transporting polypeptide 1B1; OATP1B3, organic anion transporting polypeptide 1B3; OATP2B1, organic anion transporting polypeptide 2B1; OCT4, octamer-binding transcription factor 4; OP, octylphenol; p130Cas, Crk-associated substrate; PARP, poly (ADP-ribose) polymerase; PBDEs, polybrominated diphenyl ethers; PCNB, percutaneous core needle biopsy; P-gp, permeability glycoprotein; P-MLC20, phosphorylated myosin regulatory light chain; PPT1, palmitoyl protein thioesterase-1; PXR, pregnane X receptor; Q-FISH, quantitative fl uorescence in situ hybridization; ROS, reactive oxygen species; SDF2, stromal cell-derived factor 2; SOD-2, superoxide dismutase-2; SOX2, sex determining region Y-box 2; SOX7, SRY-related HMG-box 7; SSEA4, stage-specifi c embryonic antigen-4; SUPER, sub-urothelial polyp enucleation resection; RT-PCR, reverse transcription PCR; RUT, rapid urease test; Tbdn, Tubedown; TCF7L2, transcription factor 7-like 2; TNF, tumor necrosis factor; UAAC, urothelial auto-augmentation cystoplasty; UGT1A1, UDP-glucuronosyltransferase 1A1; VLC, very long chain fatty acids. a Defi ned as regraft or, in the absence of regraft, a cloudy cornea in which there was loss of central graft clarity suffi cient to compromise vision for a minimum of 3 consecutive months.…”

“…The RIN can range from undetectable to 10, with undetectable being completely degraded and 10 being mostly intact RNA 233 ; RIN values ≤6 are usually considered insufficient for RNA studies. 210 RNA integrity can be influenced by degradation, tissue type, BMI, and by other tissue quality markers, including pH, PMI, agonal factors, and relative humidity of the processing laboratory. [230][231][232][233] In some tissues, RNA degradation increases with longer PMIs.…”

“…230 RNA preservation may be useful in RNA expression studies of individual subjects due to possible divergence of RIN and PMI values, as well as regional tissue differences in RIN. 210 Housekeeping genes must be carefully chosen and monitored to obtain normalization.…”