Current sequencing-based spatial transcriptomic methods have been widely used to resolve gene expression; however, these methods are limited to fresh or fresh-frozen samples due to the low efficiency of oligo(dT) primers in capturing RNA transcripts in degraded samples. Here, we have developed a random primer-based spatial total RNA sequencing (spRandom-seq) technology for capturing full-length total RNAs in partially degraded (low RIN values) formalin-fixed paraffin-embedded (FFPE) tissues. Our spRandom-seq approach exhibited no discernible biases at the 3'-or 5'-ends within gene bodies and could be integrated with the commercially available 10X Visium and other well-established sequencing-based spatial transcriptomic platforms. Our spRandom-seq approach outperformed oligo(dT)-based 10X Visium with an 8-fold higher capturing rate for long-non-coding RNA (lncRNA) and other non-polyadenylated RNA biotypes, including miRNA, snRNA, and miscRNA. The unsupervised clustering with solely non-coding RNAs (ncRNAs) resulted in region-specific clusters aligned perfectly with mouse brain anatomic regions. Furthermore, in the clinical FFPE sections of breast cancer, our study revealed distinct expression patterns of MUCL1 and JCHAIN within the tumor region, highlighting the inherent heterogeneity. spRandom-seq provided a potential spatial method for clinical FFPE specimens with well-established and commercially available spatial platforms, ensuring ease of operation and commercial scalability for large-scale applications.
High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples remains challenging. Here, we present hmC-CATCH, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. hmC-CATCH is based on selective 5hmC oxidation, chemical labeling and subsequent C-to-T transition during PCR. Requiring only nanoscale input genomic DNA samples, hmC-CATCH enabled us to detect genome-wide hydroxymethylome of human embryonic stem cells in a cost-effective manner. Further application of hmC-CATCH to cell-free DNA (cfDNA) of healthy donors and cancer patients revealed base-resolution hydroxymethylome in the human cfDNA for the first time. We anticipate that our chemical biology approach will find broad applications in hydroxymethylome analysis of limited biological and clinical samples.
Abstract Papillary thyroid carcinoma represents the most prevalent form of thyroid cancer, exhibiting the lowest degree of malignancy. It constitutes approximately 85% of all thyroid cancer cases. This cancer type can manifest at any age but is predominantly observed in children or young women under the age of 40. The aberrant expression of long non-coding RNA (lncRNA) and messenger RNA (mRNA) has been identified as a crucial factor in the pathogenesis of this disease. Our analysis of existing databases revealed that lncRNA GUSBP11 and the membrane progesterone receptor PAQR7 are significantly overexpressed in thyroid cancer, demonstrating a notable positive correlation between them. However, RNA sequencing analysis indicated the absence of a direct relationship between these molecules. Through a comprehensive pooled analysis across multiple studies, we discovered a direct link between miR-28-5p and both lncRNA GUSBP11 and mRNA PAQR7, which were found to be significantly downregulated in thyroid cancer cases. This research endeavors to lay a novel foundation for the development of targeted therapy for thyroid cancer, focusing on the interaction between lncRNA, miRNA, and mRNA.
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