Evaluation of DNAmAge in paired fresh, frozen, and formalin-fixed paraffin-embedded heart tissues.

The continued development in methylome analysis has enabled a more precise assessment of DNA methylation, but treatment of target tissue prior to analysis may affect DNA analysis. Prediction of age based on methylation levels in the genome (DNAmAge) has gained much interest in disease predisposition (biological age estimation), but also in chronological donor age estimation in crime case samples. Various epigenetic clocks were designed to predict the age. However, it remains unknown how the storage of the tissues affects the DNAmAge estimation. In this study, we investigated the storage method impact of DNAmAge by the comparing the DNAmAge of the two commonly used storage methods, freezing and formalin-fixation and paraffin-embedding (FFPE) to DNAmAge of fresh tissue. This was carried out by comparing paired heart tissue samples of fresh tissue, samples stored by freezing and FFPE to chronological age and whole blood samples from the same individuals. Illumina EPIC beadchip array was used for methylation analysis and the DNAmAge was evaluated with the following epigenetic clocks: Horvath, Hannum, Levine, Horvath skin+blood clock (Horvath2), PedBE, Wu, BLUP, EN, and TL. We observed differences in DNAmAge among the storage conditions. FFPE samples showed a lower DNAmAge compared to that of frozen and fresh samples. Additionally, the DNAmAge of the heart tissue was lower than that of the whole blood and the chronological age. This highlights caution when evaluating DNAmAge for FFPE samples as the results were underestimated compared with fresh and frozen tissue samples. Furthermore, the study also emphasizes the need for a DNAmAge model based on heart tissue samples for an accurate age estimation.

Improved Preservation of Mouse Intestinal Tissue Using a Formalin/Acetic Acid Fixative and Quantitative Histological Analysis Using QuPath.

The architecture and morphology of the intestinal tissue from mice or other small animals are difficult to preserve for histological and molecular analysis due to the fragile nature of this tissue. The intestinal mucosa consists of villi and crypts lined with epithelial cells. In between the epithelial folds extends the lamina propria, a loose connective tissue that contains blood and lymph vessels, fibroblasts, and immune cells. Underneath the mucosa are two layers of contractile smooth muscle and nerves. The tissue experiences significant changes during fixation, which can impair the reliability of histologic analysis. Poor-quality histologic sections are not suitable for quantitative image-based tissue analysis. This article offers a new fixative composed of neutral buffered formalin (NBF) and acetic acid, called FA. This fixative significantly improved the histology of mouse intestinal tissue compared to traditional NBF and enabled precise, reproducible histologic molecular analyses using QuPath software. Algorithmic training of QuPath allows for automated segmentation of intestinal compartments, which can be further interrogated for cellular composition and disease-related changes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Improved preservation of mouse intestinal tissue using a formalin/acetic acid fixative Support Protocol: Quantitative tissue analysis using QuPath.

Revealing intact neuronal circuitry in centimeter-sized formalin-fixed paraffin-embedded brain.

Tissue-clearing and labeling techniques have revolutionized brain-wide imaging and analysis, yet their application to clinical formalin-fixed paraffin-embedded (FFPE) blocks remains challenging. We introduce HIF-Clear, a novel method for efficiently clearing and labeling centimeter-thick FFPE specimens using elevated temperature and concentrated detergents. HIF-Clear with multi-round immunolabeling reveals neuron circuitry regulating multiple neurotransmitter systems in a whole FFPE mouse brain and is able to be used as the evaluation of disease treatment efficiency. HIF-Clear also supports expansion microscopy and can be performed on a non-sectioned 15-year-old FFPE specimen, as well as a 3-month formalin-fixed mouse brain. Thus, HIF-Clear represents a feasible approach for researching archived FFPE specimens for future neuroscientific and 3D neuropathological analyses.

A Hitchhiker's guide to high-dimensional tissue imaging with multiplexed ion beam imaging.

Advancements in multiplexed tissue imaging technologies are vital in shaping our understanding of tissue microenvironmental influences in disease contexts. These technologies now allow us to relate the phenotype of individual cells to their higher-order roles in tissue organization and function. Multiplexed Ion Beam Imaging (MIBI) is one of such technologies, which uses metal isotope-labeled antibodies and secondary ion mass spectrometry (SIMS) to image more than 40 protein markers simultaneously within a single tissue section. Here, we describe an optimized MIBI workflow for high-plex analysis of Formalin-Fixed Paraffin-Embedded (FFPE) tissues following antigen retrieval, metal isotope-conjugated antibody staining, imaging using the MIBI instrument, and subsequent data processing and analysis. While this workflow is focused on imaging human FFPE samples using the MIBI, this workflow can be easily extended to model systems, biological questions, and multiplexed imaging modalities.

DNA flow cytometry for detection of genomic instability as a cancer precursor in the gastrointestinal tract.

One of the earliest applications of flow cytometry was the measurement of DNA content in cells. This method is based on the ability to stain DNA in a stoichiometric manner (i.e., the amount of stain is directly proportional to the amount of DNA within the cell). For more than 40years, a number of studies have consistently demonstrated the utility of DNA flow cytometry as a potential diagnostic and/or prognostic tool in patients with most epithelial tumors, including pre-invasive lesions (such as dysplasia) in the gastrointestinal tract. However, its availability as a clinical test has been limited to few medical centers due to the requirement for fresh tissue in earlier studies and perceived technical demands. However, more recent studies have successfully utilized formalin-fixed paraffin-embedded (FFPE) tissue to generate high-quality DNA content histograms, demonstrating the feasibility of this methodology. This review summarizes step-by-step methods on how to perform DNA flow cytometry using FFPE tissue and analyze DNA content histograms based on the published consensus guidelines in order to assist in the diagnosis and/or risk stratification of many different epithelial tumors, with particular emphasis on dysplasia associated with Barrett's esophagus and inflammatory bowel disease.

Oil blotting paper for formalin fixation increases endoscopic ultrasound-guided tissue acquisition-collected sample volumes on glass slides.

OBJECTIVES: Endoscopic ultrasound-guided tissue acquisition (EUS-TA) is used for pathological diagnosis and obtaining samples for molecular testing, facilitating the initiation of targeted therapies in patients with pancreatic cancer. However, samples obtained via EUS-TA are often insufficient, requiring more efforts to improve sampling adequacy for molecular testing. Therefore, this study investigated the use of oil blotting paper for formalin fixation of samples obtained via EUS-TA. METHODS: This prospective study enrolled 42 patients who underwent EUS-TA for pancreatic cancer between September 2020 and February 2022 at the Osaka International Cancer Institute. After a portion of each sample obtained via EUS-TA was separated for routine histological evaluation, the residual samples were divided into filter paper and oil blotting paper groups for analysis. Accordingly, filter paper and oil blotting paper were used for the formalin fixation process. The total tissue, nuclear, and cytoplasm areas of each sample were quantitatively evaluated using virtual slides, and the specimen volume and histological diagnosis of each sample were evaluated by an expert pathologist. RESULTS: All cases were cytologically diagnosed as adenocarcinoma. The area ratios of the total tissue, nuclear, and cytoplasmic portions were significantly larger in the oil blotting paper group than in the filter paper group. The frequency of cases with large amount of tumor cells was significantly higher in the oil blotting paper group (33.3%) than in the filter paper group (11.9%) (p = 0.035). CONCLUSIONS: Oil blotting paper can increase the sample volume obtained via EUS-TA on glass slides and improve sampling adequacy for molecular testing.

Double Chromogen-based Immunohistochemical Staining: An Efficient Approach for Utilizing Long-term Formalin-fixed Tissue in Biobanks.

The New South Wales Brain Tissue Resource Centre is a human brain bank that provides top-quality brain tissue for cutting-edge neuroscience research spanning various conditions from alcohol use disorder to neurodegenerative diseases. However, the conventional practice of preserving brain tissue in formalin poses challenges for immunofluorescent staining primarily due to the formalin's tendency, over time, to create cross-links between antigens, which can obscure epitopes of interest. In addition, researchers can encounter issues such as spectral bleeding, limitations in using multiple colors, autofluorescence, and cross-reactivity when working with long-term formalin-fixed brain tissue. The purpose of the study was to test chromogen-based double immunolabeling to negate the issues with immunofluorescent staining. Colocalization of antigens was explored using chromogens 3-amino-9-ethylcarbazole (AEC) and 3,3,-diaminobenzidine in a sequential staining procedure where the AEC signal was eliminated by alcohol treatment. Combinations of 2 or 3 primary antibodies from the same or different species were trialed successfully with this protocol. The colocalization of antigens was also demonstrated with pseudocoloring that mimicked immunofluorescence staining. This staining technique increases the utility of archival formalin-fixed tissue samples.

RNA sequencing of formalin fixed paraffin-embedded heart tissue provides transcriptomic information about chemotherapy-induced cardiotoxicity.

Gene expression of formalin-fixed paraffin-embedded (FFPE) tissue may serve for molecular studies on cardiovascular diseases. Chemotherapeutics, such as doxorubicin (DOX) may cause heart injury, but the mechanisms of these side effects of DOX are not well understood. This study aimed to investigate whether DOX-induced gene expression in archival FFPE heart tissue in experimental rats would correlate with the gene expression in fresh-frozen heart tissue by applying RNA sequencing technology. The results showed RNA from FFPE samples was degraded, resulting in a lower number of uniquely mapped reads. However, DOX-induced differentially expressed genes in FFPE were related to molecular mechanisms of DOX-induced cardiotoxicity, such as inflammation, calcium binding, endothelial dysfunction, senescence, and cardiac hypertrophy signaling. Our data suggest that, despite the limitations, RNA sequencing of archival FFPE heart tissue supports utilizing FFPE tissues from retrospective studies on cardiovascular disorders, including DOX-induced cardiotoxicity.

CytoLyt fixation impedes insulinoma-associated protein 1 (INSM1) immunoreactivity compared to formalin fixation.

INTRODUCTION: Insulinoma-associated protein 1 (INSM1) is an immunohistochemical marker commonly used to confirm cytomorphological concordant neuroendocrine tumors/carcinomas (NETs/NECs), demonstrating high utility in small samples. Previous reports have suggested comparable INSM1 staining in CytoLyt-fixed cell blocks and formalin-fixed surgical pathology specimens. This study aimed to assess INSM1 immunoreactivity using both fixation methods and investigate potential factors contributing to its variable expression. MATERIALS AND METHODS: A retrospective query was performed (03/31/21-05/31/22) for NET/NEC cases that had both formalin- and CytoLyt-fixed cell blocks. We collected clinical data and reporting of immunostains for each case. INSM1 staining was evaluated in both fixation methods, and reported as positive, negative, or equivocal. Equivocal INSM1 staining was further scored as a percentage of 1%-100% and intensity of weak (faint staining), moderate (darker staining), and strong (dense staining). RESULTS: Our search identified 20 cases from diverse body sites, including mediastinal lymph nodes (40%), pancreas (35%), lung (20%), and porta hepatis lymph nodes (5%). All cases exhibited a widespread positivity (over 90%) in formalin-fixed cell blocks. In contrast, CytoLyt fixed cells showed a negative stain in 65% of cases and 30% exhibited an equivocal positivity. CONCLUSIONS: While INSM1 is previously reported as a sensitive (75%-100%) and specific (82.7%-100%) marker for NET/NECs, our study found a reduced immunohistochemical staining in CytoLyt-fixed cell blocks. Consequently, false negative INSM1 immunohistochemical results in CytoLyt-fixed cell block material may pose a pitfall in the diagnosis of NET/NEC.

Walking a thin line between fixation and epitope binding - characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue.

Antibody-based fluorescence analysis of female reproductive tissues in research of sexually transmitted diseases allows for an in-depth understanding of protein localization, interactions, and pathogenesis. However, in many cases, cryosectioning is not compatible with biosafety regulations; at all times, exposure of lab personnel and the public to potentially harmful pathogens from biological infectious material must be avoided; thus, formaldehyde fixation is essential. Due to formaldehyde's cross-linking properties, protein detection with antibodies can be impeded. To allow effective epitope binding during immunofluorescence of formalin-fixed paraffin-embedded vaginal tissue, we investigated two antigen retrieval methods. We tested these methods regarding their suitability for automated image analysis, facilitating reproducible quantitative microscopic data acquisition in sexually transmitted disease research. Heat-based retrieval at 80°C in citrate buffer proved to increase antibody binding to eosinophil protein and HSV-2 visibly and tissue morphology best, and was the most efficient for sample processing and quantitative analysis.

Procrustes is a machine-learning approach that removes cross-platform batch effects from clinical RNA sequencing data.

With the increased use of gene expression profiling for personalized oncology, optimized RNA sequencing (RNA-seq) protocols and algorithms are necessary to provide comparable expression measurements between exome capture (EC)-based and poly-A RNA-seq. Here, we developed and optimized an EC-based protocol for processing formalin-fixed, paraffin-embedded samples and a machine-learning algorithm, Procrustes, to overcome batch effects across RNA-seq data obtained using different sample preparation protocols like EC-based or poly-A RNA-seq protocols. Applying Procrustes to samples processed using EC and poly-A RNA-seq protocols showed the expression of 61% of genes (N = 20,062) to correlate across both protocols (concordance correlation coefficient > 0.8, versus 26% before transformation by Procrustes), including 84% of cancer-specific and cancer microenvironment-related genes (versus 36% before applying Procrustes; N = 1,438). Benchmarking analyses also showed Procrustes to outperform other batch correction methods. Finally, we showed that Procrustes can project RNA-seq data for a single sample to a larger cohort of RNA-seq data. Future application of Procrustes will enable direct gene expression analysis for single tumor samples to support gene expression-based treatment decisions.

FixNCut: single-cell genomics through reversible tissue fixation and dissociation.

The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.

Extracting high-quality RNA from formaldehyde-fixed naturally aged neuromusculoskeletal tissues.

Modern approaches to discovering molecular mechanisms and validating treatments for age-related neuromusculoskeletal dysfunction typically rely on high-throughput transcriptome analysis. Previously harvested and fixed tissues offer an incredible reservoir of untapped molecular information. However, obtaining RNA from such formaldehyde-fixed neuromusculoskeletal tissues, especially fibrotic aged tissues, is technically challenging and often results in RNA degradation, chemical modification and yield reduction, prohibiting further analysis. Therefore, we developed a protocol to extract high-quality RNA from formaldehyde-fixed brain, cartilage, muscle and peripheral nerve isolated from naturally aged mice. Isolated RNA produced reliable gene expression data comparable to fresh and flash-frozen tissues and was sensitive enough to detect age-related changes, making our protocol valuable to researchers in the field of aging.

Systematic development of immunohistochemistry protocol for large cryosections-specific to non-perfused fetal brain.

BACKGROUND: Immunohistochemistry (IHC) is an important technique in understanding the expression of neurochemical molecules in the developing human brain. Despite its routine application in the research and clinical setup, the IHC protocol specific for soft fragile fetal brains that are fixed using the non-perfusion method is still limited in studying the whole brain. NEW METHOD: This study shows that the IHC protocols, using a chromogenic detection system, used in animals and adult humans are not optimal in the fetal brains. We have optimized key steps from Antigen retrieval (AR) to chromogen visualization for formalin-fixed whole-brain cryosections (20 µm) mounted on glass slides. RESULTS: We show the results from six validated, commonly used antibodies to study the fetal brain. We achieved optimal antigen retrieval with 0.1 M Boric Acid, pH 9.0 at 70°C for 20 minutes. We also present the optimal incubation duration and temperature for protein blocking and the primary antibody that results in specific antigen labeling with minimal tissue damage. COMPARISON WITH EXISTING METHODS: The IHC protocol commonly used for adult human and animal brains results in significant tissue damage in the fetal brains with little or suboptimal antigen expression. Our new method with important modifications including the temperature, duration, and choice of the alkaline buffer for AR addresses these pitfalls and provides high-quality results. CONCLUSION: The optimized IHC protocol for the developing human brain (13-22 GW) provides a high-quality, repeatable, and reliable method for studying chemoarchitecture in neurotypical and pathological conditions across different gestational ages.

Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling.

Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

DEEPOMICS FFPE, a deep neural network model, identifies DNA sequencing artifacts from formalin fixed paraffin embedded tissue with high accuracy.

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens are routinely used in pathological diagnosis, but their large number of artifactual mutations complicate the evaluation of companion diagnostics and analysis of next-generation sequencing data. Identification of variants with low allele frequencies is challenging because existing FFPE filtering tools label all low-frequency variants as artifacts. To address this problem, we aimed to develop DEEPOMICS FFPE, an AI model that can classify a true variant from an artifact. Paired whole exome sequencing data from fresh frozen and FFPE samples from 24 tumors were obtained from public sources and used as training and validation sets at a ratio of 7:3. A deep neural network model with three hidden layers was trained with input features using outputs of the MuTect2 caller. Contributing features were identified using the SHapley Additive exPlanations algorithm and optimized based on training results. The performance of the final model (DEEPOMICS FFPE) was compared with those of existing models (MuTect filter, FFPolish, and SOBDetector) by using well-defined test datasets. We found 41 discriminating properties for FFPE artifacts. Optimization of property quantification improved the model performance. DEEPOMICS FFPE removed 99.6% of artifacts while maintaining 87.1% of true variants, with an F1-score of 88.3 in the entire dataset not used for training, which is significantly higher than those of existing tools. Its performance was maintained even for low-allele-fraction variants with a specificity of 0.995, suggesting that it can be used to identify subclonal variants. Different from existing methods, DEEPOMICS FFPE identified most of the sequencing artifacts in the FFPE samples while retaining more of true variants, including those of low allele frequencies. The newly developed tool DEEPOMICS FFPE may be useful in designing capture panels for personalized circulating tumor DNA assay and identifying candidate neoepitopes for personalized vaccine design. DEEPOMICS FFPE is freely available on the web ( http://deepomics.co.kr/ffpe ) for research.

Effects of autolysis and prolonged formalin fixation on histomorphology and immunohistochemistry of normal canine brain tissue: an experimental study.

CNS tumor diagnosis in dogs often relies on immunohistochemistry (IHC) given similar histologic features among tumors. Most CNS tissue samples encountered by diagnostic pathologists are collected during autopsy, and postmortem specimens can be susceptible to autolysis and prolonged formalin fixation, both of which have the potential to influence IHC results and interpretation. Here we evaluated the effects of experimentally controlled autolysis induced by delayed tissue fixation (sections of brain held for 2, 4, 8, 12, 24, 48, and 72 h in 0.9% NaCl at either room temperature or 37°C prior to fixation) as well as the effects of prolonged formalin fixation times (1 wk, 1 mo, 2 mo) on a panel of 8 IHC markers (CNPase, GFAP, Iba1, OLIG2, PGP9.5, MAP2, NeuN, synaptophysin) relevant to brain tumor diagnosis. Prolonged fixation of up to 2 mo had no detrimental effect on any immunomarker except NeuN, which had reduced immunolabeling intensity. Delayed fixation led to autolytic changes as expected, on a gradient of severity corresponding to increased time in saline prior to fixation. Several immunomarkers should be used with caution (CNPase, OLIG2) or avoided entirely (MAP2, NeuN) in markedly autolyzed brain and brain tumor tissues. Our results suggest that autolysis has minimal effect on most immunomarkers, but that advanced autolysis may cause a loss of specificity for GFAP, MAP2, and PGP9.5, a loss of intensity of CNPase and OLIG2, and loss of labeling with MAP2 and NeuN. Prolonged fixation affected only NeuN, with mildly decreased intensity.

High-Resolution Genotyping of Formalin-Fixed Tissue Accurately Estimates Polygenic Risk Scores in Human Diseases.

Formalin-fixed paraffin-embedded (FFPE) tissues stored in biobanks and pathology archives are a vast but underutilized source for molecular studies on different diseases. Beyond being the "gold standard" for preservation of diagnostic human tissues, FFPE samples retain similar genetic information as matching blood samples, which could make FFPE samples an ideal resource for genomic analysis. However, research on this resource has been hindered by the perception that DNA extracted from FFPE samples is of poor quality. Here, we show that germline disease-predisposing variants and polygenic risk scores (PRS) can be identified from FFPE normal tissue (FFPE-NT) DNA with high accuracy. We optimized the performance of FFPE-NT DNA on a genome-wide array containing 657,675 variants. Via a series of testing and validation phases, we established a protocol for FFPE-NT genotyping with results comparable with blood genotyping. The median call rate of FFPE-NT samples in the validation phase was 99.85% (range 98.26%-99.94%) and median concordance with matching blood samples was 99.79% (range 98.85%-99.9%). We also demonstrated that a rare pathogenic PALB2 genetic variant predisposing to cancer can be correctly identified in FFPE-NT samples. We further imputed the FFPE-NT genotype data and calculated the FFPE-NT genome-wide PRS in 3 diseases and 4 disease risk variables. In all cases, FFPE-NT and matching blood PRS were highly concordant (all Pearson's r > 0.95). The ability to precisely genotype FFPE-NT on a genome-wide array enables translational genomics applications of archived FFPE-NT samples with the possibility to link to corresponding phenotypes and longitudinal health data.

Canine urothelial carcinoma: a pilot study of microRNA detection in formalin-fixed, paraffin-embedded tissue samples and in normal urine.

We assessed the effects of fixation time in formalin and inclusion of surrounding tissue on microRNA (miRNA) cycle quantification (Cq) values in formalin-fixed, paraffin-embedded (FFPE) urothelial carcinoma (UC) tissue (n = 3), and the effect of conditions on miRNAs in urine from 1 healthy dog. MiRNAs were extracted using commercial kits and quantified using miRNA-specific fluorometry in normal bladder tissue scrolls, UC tissue cores, and bladder muscularis tissue cores from 4 FFPE bladder sections (3 UCs, 1 normal), plus 1 UC stored in formalin for 1, 8, 15, and 22 d before paraffin-embedding. Urine was collected from a healthy dog on 4 occasions; 1-mL aliquots were stored at 20, 4, -20, and -80°C for 4, 8, 24, and 48 h, and 1 and 2 wk. For both FFPE tissue and urine, we used reverse-transcription quantitative real-time PCR (RT-qPCR) to quantify miR-143, miR-152, miR-181a, miR-214, miR-1842, and RNU6B in each tissue or sample, using miR-39 as an exogenous control gene. The Cq values were compared with ANOVA and t-tests. The time of tissue-fixation in formalin did not alter miRNA Cq values; inclusion of the muscularis layer resulted in a statistically different miRNA Cq profile for miR-152, miR-181a, and RNU6B in bladder tissue. MiRNAs in acellular urine were stable for up to 2 wk regardless of the storage temperature. Our findings support using stored FFPE and urine samples for miRNA detection; we recommend measuring miRNA only in the tissue of interest in FFPE sections.

Construction of multiplexed transcriptome NGS libraries of microdissected tissue samples based on combinational DNA barcode microbeads.

The combination of single-cell RNA sequencing and microdissection techniques that preserves positional information has become a major tool for spatial transcriptome analyses. However, high costs and time requirements, especially for experiments at the single cell scale, make it challenging for this approach to meet the demand for increased throughput. Therefore, we proposed combinational DNA barcode (CDB)-seq as a medium-throughput, multiplexed approach combining Smart-3SEQ and CDB magnetic microbeads for transcriptome analyses of microdissected tissue samples. We conducted a comprehensive comparison of conditions for CDB microbead preparation and related factors and then applied CDB-seq to RNA extracts, fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) mouse brain tissue samples. CDB-seq transcriptomic profiles of tens of microdissected samples could be obtained in a simple, cost-effective way, providing a promising method for future spatial transcriptomics.