LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue.

Whole transcriptome analysis (WTA) using RNA extracted from Formalin Fixed Paraffin Embedded (FFPE) tissue is an invaluable tool to understand the molecular pathology of disease. RNA extracted from FFPE tissue is either degraded and/or in very low quantities hampering gene expression analysis. Earlier studies described protocols applied for cellular RNA using poly-A primer-based linear amplification. The current study describes a method, LINCATRA (LINear amplifiCAtion of RNA for whole TRAnscriptome analysis). It employs random nonamer primer based method which can amplify short, fragmented RNA with high fidelity from as low as 5 ng to obtain enough material for WTA. The two-cycle method significantly amplified RNA at ∼1000 folds (p < 0.0001) improving the mean read lengths (p < 0.05) in WTA. Overall, increased mean read length positively correlated with on-target reads (Pearson's r = 0.71, p < 0.0001) in both amplified and unamplified RNA-seq analysis. Gene expression analysis compared between unamplified and amplified group displayed substantial overlap of the differentially expressed genes (DEGs) (log2 fold change cut-off < -2 and >2, p < 0.05) identified between lung cancer and asthma cohorts validating the method developed. This method is applicable in clinical molecular pathology field for both diagnostics and elucidation of disease mechanisms.

A Fast, Affordable, and Minimally Invasive Diagnostic Test for Cancer of Unknown Primary Using DNA Methylation Profiling.

Currently, we cannot provide a conclusive diagnosis for 3% to 5% of people who are confronted with cancer. These patients have cancer of unknown primary (CUP), ie, a metastasized cancer for which the tissue of origin cannot be determined. Studies have shown that the DNA methylation profile is a unique "fingerprint" that can be used to classify tumors. Here we used cell-free reduced representation bisulfite sequencing (cfRRBS), a technique that allows us to identify the methylation profile starting from minimal amounts of highly fragmented DNA, for CUP diagnosis on formalin-fixed paraffin-embedded (FFPE) tissue and liquid biopsies. We collected 80 primary tumor FFPE samples covering 16 tumor entities together with 15 healthy plasma samples to use as a custom cfRRBS reference data set. Entity-specific methylation regions are defined for each entity to build a classifier based on nonnegative least squares deconvolution. This classification framework was tested on 30 FFPE, 19 plasma, and 40 pleural and peritoneal effusion samples of both known metastatic tumors and clinical CUPs for which pathological investigation finally resulted in a cancer diagnosis. Using this framework, 27 of 30 FFPE (all CUPs) and 16 of 19 plasma samples (10/13 CUPs) obtained an accurate diagnosis, with a minimal DNA input of 400 pg. Diagnosis of the 40 pleural and peritoneal effusion samples is possible in 9 of 27 samples with negative/inconclusive cytology (6/13 CUPs), showing that cell-free DNA (cfDNA) methylation profiling could complement routine cytologic analysis. However, a low "cfDNA - high-molecular weight DNA ratio" has a considerable impact on the prediction accuracy. Moreover, the accuracy improves significantly if the predicted tumor percentage is >7%. This proof-of-concept study shows the feasibility of using DNA methylation profiling on FFPE and liquid biopsy samples such as blood, ascites, and pleural effusions in a fast and affordable way. Our novel RRBS-based technique requires minimal DNA input, can be performed in

Microsatellite Instability Testing and Prognostic Implications in Colorectal Cancer.

Given the crucial predictive implications of microsatellite instability (MSI) in colorectal cancer (CRC), MSI screening is commonly performed in those with and at risk for CRC. Here, we compared results from immunohistochemistry (IHC) and the droplet digital PCR (ddPCR) MSI assay on formalin-fixed paraffin-embedded tumor samples from 48 patients who underwent surgery for colon and rectal cancer by calculating Cohen's kappa measurement (k), revealing high agreement between the methods (k = 0.915). We performed Kaplan-Meier survival analyses and univariate and multivariate Cox regression to assess the prognostic significance of ddPCR-based MSI and to identify clinicopathological features associated with CRC outcome. Patients with MSI-high had better overall survival (OS; p = 0.038) and disease-free survival (DFS; p = 0.049) than those with microsatellite stability (MSS). When stratified by primary tumor location, right-sided CRC patients with MSI-high showed improved DFS, relative to those with MSS (p < 0.001), but left-sided CRC patients did not. In multivariate analyses, MSI-high was associated with improved OS (hazard ratio (HR) = 0.221, 95% confidence interval (CI): 0.026-0.870, p = 0.042), whereas the loss of DNA mismatch repair protein MutL homolog 1 (MLH1) expression was associated with worse OS (HR = 0.133, 95% CI: 0.001-1.152, p = 0.049). Our results suggest ddPCR is a promising tool for MSI detection. Given the opposing effects of MSI-high and MLH1 loss on OS, both ddPCR and IHC may be complementary for the prognostic assessment of CRC.

Multitarget Fluorescence In Situ Hybridization Diagnostic Applications in Tumors.

Multitarget fluorescence in situ hybridization (mFISH) is a technique that allows the detection of multiple target sequences on the same sample using spectrally distinct fluorophore labels. The mFISH approach is currently a useful assay in the oncologic field for the detection of predictive, prognostic, and diagnostic biomarkers. In this chapter, we summarize the application of mFISH in the identification of target genetic aberrations in formalin-fixed, paraffin-embedded (FFPE) tissue samples of several tumor types. We discuss the mFISH protocols in FFPE samples, the innovative multitarget probes used, and the critical issues related to their interpretation.

Quantifying Protein Acetylation in Diabetic Nephropathy from Formalin-Fixed Paraffin-Embedded Tissue.

PURPOSE: Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus and a leading cause of chronic kidney disease and end-stage renal disease. One potential mechanism underlying cellular dysfunction contributing to kidney disease is aberrant protein post-translational modifications. Lysine acetylation is associated with cellular metabolic flux and is thought to be altered in patients with diabetes and dysfunctional renal metabolism. EXPERIMENTAL DESIGN: A novel extraction and LC-MS/MS approach was adapted to quantify sites of lysine acetylation from formalin-fixed paraffin-embedded (FFPE) kidney tissue and from patients with DKD and non-diabetic donors (n = 5 and n = 7, respectively). RESULTS: Analysis of FFPE tissues identified 840 total proteins, with 225 of those significantly changing in patients with DKD. Acetylomic analysis quantified 289 acetylated peptides, with 69 of those significantly changing. Pathways impacted in DKD patients revealed numerous metabolic pathways, specifically mitochondrial function, oxidative phosphorylation, and sirtuin signaling. Differential protein acetylation in DKD patients impacted sirtuin signaling, valine, leucine, and isoleucine degradation, lactate metabolism, oxidative phosphorylation, and ketogenesis. CONCLUSIONS AND CLINICAL RELEVANCE: A quantitative acetylomics platform was developed for protein biomarker discovery in formalin-fixed and paraffin-embedded biopsies of kidney transplant patients suffering from DKD.

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.

Genome-wide somatic mutation analysis of sinonasal adenocarcinoma with and without wood dust exposure.

BACKGROUND: Sinonasal adenocarcinoma is a rare cancer, encompassing two different entities, the intestinal-type sinonasal adenocarcinoma (ITAC) and the non-intestinal-type sinonasal adenocarcinoma (non-ITAC). Occurrence of ITAC is strongly associated with exposure to hardwood dusts. In countries with predominant exposure to softwood dust the occurrence of sinonasal adenocarcinomas is lower and the relative amount of non-ITACs to ITACs is higher. The molecular mechanisms behind the tumorigenic effects of wood dust remain largely unknown. METHODS: We carried out whole-genome sequencing of formalin-fixed paraffin-embedded (FFPE) samples of sinonasal adenocarcinomas from ten wood dust-exposed and six non-exposed individuals, with partial tobacco exposure data. Sequences were analyzed for the presence of mutational signatures matching COSMIC database signatures. Driver mutations and CN variant regions were characterized. RESULTS: Mutation burden was higher in samples of wood dust-exposed patients (p = 0.016). Reactive oxygen species (ROS) damage-related mutational signatures were almost exclusively identified in ITAC subtype samples (p = 0.00055). Tobacco smoke mutational signatures were observed in samples of patients with tobacco exposure or missing information, but not in samples from non-exposed patients. A tetraploidy copy number (CN) signature was enriched in ITAC subtype (p = 0.042). CN variation included recurrent gains in COSMIC Cancer Gene Census genes TERT, SDHA, RAC1, ETV1, PCM1, and MYC. Pathogenic variants were observed most frequently in TP53, NF1, CHD2, BRAF, APC, and LRP1B. Driver mutations and copy number gains did not segregate by subtype. CONCLUSIONS: Our analysis identified distinct mutational characteristics in ITAC and non-ITAC. Mutational signature analysis may eventually become useful for documentation of occupation-related cancer, while the exact mechanisms behind wood dust-driven carcinogenesis remain elusive. The presence of homologous recombination deficiency signatures implies a novel opportunity for treatment, but further studies are needed.

snRNAs from Radical Prostatectomy Specimens Have the Potential to Serve as Prognostic Factors for Clinical Recurrence after Biochemical Recurrence in Patients with High-Risk Prostate Cancer.

In patients with high-risk prostate cancer (HRPC) after radical prostatectomy (RP), biochemical recurrence (BCR) increases the risk of distant metastasis. Accordingly, additional prognostic biomarkers are required to identify the subpopulation of patients with HRPC who develop clinical recurrence (CR) after BCR. The objective of this study was to identify biomarkers in formalin-fixed paraffin-embedded (FFPE) RP samples that are prognostic for CR in patients with HRPC who experience BCR after RP (post-RP BCR). First, we performed a preliminary RNA sequencing analysis to comprehensively profile RNA expression in FFPE RP samples obtained from patients with HRPC who developed CR after post-RP BCR and found that many snRNAs were very abundant in preserved FFPE samples. Subsequently, we used quantitative polymerase chain reaction (qPCR) to compare the expression levels of highly abundant snRNAs in FFPE RP samples from patients with HRPC with and without CR after post-RP BCR (21 CR patients and 46 non-CR patients who had more than 5 years of follow-up after BCR). The qPCR analysis revealed that the expression levels of snRNA RNU1-1/1-2 and RNU4-1 were significantly higher in patients with CR than in patients without CR. These snRNAs were significantly correlated with clinical recurrence-free survival (RFS) in patients with HRPC who experienced post-RP BCR. Furthermore, snRNA RNU1-1/1-2 could serve as an independent prognostic factor for clinical RFS in post-RP BCR of HRPC cases where known prognostic factors (e.g., Gleason score) cannot distinguish between CR and non-CR patients. Our findings provide new insights into the involvement of snRNAs in prostate cancer progression.

In Situ Hybridization of Brain Slices.

In situ hybridization (ISH) is an important technique for identifying gene expression at the cellular level in various organs, including brain slices. This approach hybridizes nucleic acid probes to cellular mRNA, allowing the detection of transcriptional products. Recent advances have enabled RNA preservation in formalin-fixed paraffin-embedded (FFPE) samples, making ISH applicable to brain tumor diagnosis and research. Here, we provide a concise overview of the standard application of chromogenic ISH in neuroscience research and neuropathology practice using FFPE blocks of brain slice sections.

Immunohistochemistry of Brain Tissues.

Immunohistochemistry (IHC) is the basis of histological or pathological analysis and is widely used to enable the detection and characterization of proteins in various organ tissues, including brain tissues. IHC is commonly performed on formalin-fixed paraffin-embedded (FFPE) tissues because of their easy storage and versatility. IHC is a key method for providing more accurate analysis of localization and function of neurons, neuroendocrine cells, and neural stem cells in the brain and other nervous systems. The related cells such as glial cells and neurovascular units have also been analyzed by IHC. Visualization of antibody-antigen interactions can be performed primarily in one of the following ways: chromogenically stained IHC and fluorescently stained IHC. In chromogenically stained IHC, an antibody is chemically conjugated to an enzyme, such as peroxidase, that can be reacted with a suitable substrate to give a colored product. In fluorescently stained IHC, the antibodies are finally tagged with fluorescent chemicals such as fluorescein isothiocyanate (FITC) or rhodamine. Here, we describe the standard methods of IHC applied to brain slice sections. Furthermore, an automated immunostainer is presented as another option for standardized immunohistochemistry.

Case report: Cutaneous anthrax diagnosed using mNGS of a formalin-fixed paraffin-embedded tissue sample.

The metagenomic next-generation sequencing (mNGS) method is preferred for genotyping useful for the identification of organisms, illumination of metabolic pathways, and determination of microbiota. It can accurately obtain all the nucleic acid information in the test sample. Anthrax is one of the most important zoonotic diseases, infecting mainly herbivores and occasionally humans. The disease has four typical clinical forms, cutaneous, gastrointestinal, inhalation, and injection, all of which may result in sepsis or meningitis, with cutaneous being the most common form. Here, we report a case of cutaneous anthrax diagnosed by mNGS in a butcher. Histopathology of a skin biopsy revealed PAS-positive bacilli. Formalin-fixed paraffin-embedded (FFPE) tissue sample was confirmed the diagnosis of anthrax by mNGS. He was cured with intravenous penicillin. To our knowledge, this is the first case of cutaneous anthrax diagnosed by mNGS using FFPE tissue. mNGS is useful for identifying pathogens that are difficult to diagnose with conventional methods, and FFPE samples are simple to manage. Compared with traditional bacterial culture, which is difficult to cultivate and takes a long time, mNGS can quickly and accurately help us diagnose anthrax, so that anthrax can be controlled in a timely manner and prevent the outbreak of epidemic events.

Comprehensive analysis of human tissues reveals unique expression and localization patterns of HSF1 and HSF2.

Heat shock factors (HSFs) are the main transcriptional regulators of the evolutionarily conserved heat shock response. Beyond cell stress, several studies have demonstrated that HSFs also contribute to a vast variety of human pathologies, ranging from metabolic diseases to cancer and neurodegeneration. Despite their evident role in mitigating cellular perturbations, the functions of HSF1 and HSF2 in physiological proteostasis have remained inconclusive. Here, we analyzed a comprehensive selection of paraffin-embedded human tissue samples with immunohistochemistry. We demonstrate that both HSF1 and HSF2 display distinct expression and subcellular localization patterns in benign tissues. HSF1 localizes to the nucleus in all epithelial cell types, whereas nuclear expression of HSF2 was limited to only a few cell types, especially the spermatogonia and the urothelial umbrella cells. We observed a consistent and robust cytoplasmic expression of HSF2 across all studied smooth muscle and endothelial cells, including the smooth muscle cells surrounding the vasculature and the high endothelial venules in lymph nodes. Outstandingly, HSF2 localized specifically at cell-cell adhesion sites in a broad selection of tissue types, such as the cardiac muscle, liver, and epididymis. To the best of our knowledge, this is the first study to systematically describe the expression and localization patterns of HSF1 and HSF2 in benign human tissues. Thus, our work expands the biological landscape of these factors and creates the foundation for the identification of specific roles of HSF1 and HSF2 in normal physiological processes.

Using formalin fixed paraffin embedded tissue to characterize the microbiota in p16-positive and p16-negative tongue squamous cell carcinoma: a pilot study.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is the most common oral cavity cancer, and p16 immunohistochemistry is an exact and available tool in the prognostic and predictive characterization of squamous cell cancers in the head and neck. Microorganisms have a close relationship with the development of TSCC. However, the association between oral bacteria and p16 status has not been well defined in the case of TSCC. Compared with traditional clinical microbial collection methods, formalin-fixed paraffin-embedded (FFPE) tissue samples have several advantages. METHODS: To compare the microbiota compositions between p16-positive and p16-negative patients with TSCC, we performed a small pilot study of microbiological studies of TSCC by paraffin tissue. DNA from FFPE tissue blocks were extracted and microbiomes were profiled by sequencing the 16 S-rRNA-encoding gene (V1-V2/V3-V4/V4 regions). Alterations in the functional potential of the microbiome were predicted using PICRUSt, Tax4Fun, and BugBase. RESULTS: A total of 60 patients with TSCC were enrolled in the study, however, some challenges associated with DNA damage in FFPE tissues existed, and only 27 (15 p16-positive and 12 p16-negative) passed DNA quality control. Nevertheless, we have tentatively found some meaningful results. The p16 status is associated with microbiota diversity, which is significantly increased in p16-positive patients compared with p16-negative patients. Desulfobacteria, Limnochordia, Phycisphaerae, Anaerolineae, Saccharimonadia and Kapabacteria had higher abundances among participants with p16-positive. Moreover, functional prediction revealed that the increase of these bacteria may enhance viral carcinogenesis in p16-positive TSCC. CONCLUSIONS: Bacterial profiles showed a significant difference between p16-positive TSCC and p16-negative TSCC. These findings may provide insights into the relationship between p16 status and the microbial taxa in TSCC, and these bacteria may provide new clues for developing therapeutic targets for TSCC.

A case report of carcinoma of the papilla of Vater associated with a hyperplasia-dysplasia-carcinoma sequence by pancreaticobiliary maljunction.

BACKGROUND: Pancreaticobiliary maljunction (PBM) is a known risk factor for biliary tract cancer. However, its association with carcinoma of the papilla of Vater (PVca) remains unknown. We report a case with PVca that was thought to be caused by the hyperplasia-dysplasia-carcinoma sequence, which is considered a mechanism underlying PBM-induced biliary tract cancer. CASE PRESENTATION: A 70-year-old woman presented with white stool and had a history of cholecystectomy for the diagnosis of a non-dilated biliary tract with PBM. Esophagogastroduodenoscopy revealed a tumor in the papilla of Vater, and PVca was histologically proven by biopsy. We finally diagnosed her with PVca concurrent with non-biliary dilated PBM (cT1aN0M0, cStage IA, according to the Union for International Cancer Control, 8th edition), and subsequently performed subtotal stomach-preserving pancreaticoduodenectomy. Pathological findings of the resected specimen revealed no adenomas and dysplastic and hyperplastic mucosae in the common channel slightly upstream of the main tumor, suggesting a PBM related carcinogenic pathway with hyperplasia-dysplasia-carcinoma sequence. Immunostaining revealed positivity for CEA. CK7 positivity, CK20 negativity, and MUC2 negativity indicated that this PVca was of the pancreatobiliary type. Genetic mutations were exclusively detected in tumors and not in normal tissues, and bile ducts from formalin-fixed paraffin-embedded samples included mutated-ERBB2 (Mutant allele frequency, 81.95%). Moreover, of the cell-free deoxyribonucleic acid (cfDNA) extracted from liquid biopsy mutated-ERBB2 was considered the circulating-tumor deoxyribonucleic acid (ctDNA) of this tumor. CONCLUSIONS: Herein, we report the first case of PVca with PBM potentially caused by a "hyperplasia-dysplasia-carcinoma sequence" detected using immunostaining and next-generation sequencing. Careful follow-up is required if pancreaticobiliary reflux persists, considering the possible development of PVca.

Two complete 1918 influenza A/H1N1 pandemic virus genomes characterized by next-generation sequencing using RNA isolated from formalin-fixed, paraffin-embedded autopsy lung tissue samples along with evidence of secondary bacterial co-infection.

The 1918 influenza pandemic was the most devastating respiratory pandemic in modern human history, with 50-100 million deaths worldwide. Here, we characterized the complete genomes of influenza A virus (IAV) from two fatal cases during the fall wave of 1918 influenza A (H1N1) pandemic in the United States, one from Walter Reed Army Hospital in Washington, DC, and the other from Camp Jackson, SC. The two complete IAV genomes were obtained by combining Illumina deep sequencing data from both total RNA and influenza viral genome-enriched libraries along with Sanger sequencing data from PCR across the sequencing gaps. This study confirms the previously reported 1918 IAV genomes and increases the total number of available complete or near-complete influenza viral genomes of the 1918 pandemic from four to six. Sequence comparisons among them confirm that the genomes of the 1918 pandemic virus were highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases. Interestingly, in the Washington, DC, case, evidence is presented of the first reported Rhodococcus-influenza virus co-infection. IMPORTANCE: This study applied modern molecular biotechnology and high-throughput sequencing to formalin-fixed, paraffin-embedded autopsy lung samples from two fatal cases during the fall wave of the 1918 influenza A (H1N1) pandemic in the United States. Complete influenza genomes were obtained from both cases, which increases the total number of available complete or near-complete influenza genomes of the 1918 pandemic virus from four to six. Sequence analysis confirms that the 1918 pandemic virus was highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases, including the first reported evidence of Rhodococcus-influenza co-infection. Overall, this study offers a detailed view at the molecular level of the very limited samples from the most devastating influenza pandemic in modern human history.

LC-MS bioanalysis of protein biomarkers and protein therapeutics in formalin-fixed paraffin-embedded tissue specimens.

Formalin-fixed paraffin-embedded (FFPE) is a form of preservation and preparation for biopsy specimens. FFPE tissue specimens are readily available as part of oncology studies because they are often collected for disease diagnosis or confirmation. FFPE tissue specimens could be extremely useful for retrospective studies on protein biomarkers because the samples preserved in FFPE blocks could be stable for decades. However, LC-MS bioanalysis of FFPE tissues poses significant challenges. In this Perspective, we review the benefits and recent developments in LC-MS approach for targeted protein biomarker and protein therapeutic analysis using FFPE tissues and their clinical and translational applications. We believe that LC-MS bioanalysis of protein biomarkers in FFPE tissue specimens represents a great potential for its clinical applications.

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.

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.