Extraction-Free Whole Transcriptome Profiling from Specific Subareas of Unstained or H&E Stained FFPE Tissues

We describe the use of a ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq™, to profile formalin-fixed paraffin-embedded (FFPE) tissue, including H&E stained FFPE tissue, by directly lysing tissue scraped from slides without extracting RNA or converting the RNA to cDNA. The correlation of measured gene expression changes in unfixed and fixed samples using blocks prepared from a pellet of a single cell type was R2 = 0.97, demonstrating that no significant artifacts were introduced by fixation. Fixed and fresh samples prepared in an equivalent manner produced comparable sequencing depth results (+/- 20%), with similar %CV (11.5 and 12.7%, respectively), indicating no significant loss of measurable RNA due to fixation. The sensitivity of the TempO-Seq assay was the same whether the tissue section was fixed or not. The assay performance was equivalent for human, mouse, or rat whole transcriptome. The results from 10 mm2 and 2 mm2 areas of tissue obtained from 5 μm thick sections were equivalent, thus demonstrating high sensitivity and ability to profile focal areas of histology within a section. Replicate reproducibility of separate areas of tissue ranged from R2= 0.83 (lung) to 0.96 (liver) depending on the tissue type, with an average correlation of R2 = 0.90 across nine tissue types. The average %CV’s were 16.8% for genes expressed at greater than 200 counts, and 20.3% for genes greater than 50 counts. Tissue specific differences in gene expression were identified and agreed with the literature. There was negligible impact on assay performance using FFPE tissues that had been archived for up to 30 years. Similarly, there was negligible impact of H&E staining, facilitating accurate visualization for scraping and assay of small focal areas of specific histology within a section.本研究介绍了基于连接的靶向全转录组表达谱分析实验（ligation-based targeted whole transcriptome expression profiling assay）TempO-Seq™的应用：该实验可直接裂解从玻片上刮取的组织，无需提取RNA或将RNA反转录为cDNA，即可对福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）组织（包括经苏木精-伊红染色（hematoxylin-eosin, H&E）的FFPE组织）开展表达谱分析。以单一细胞类型沉淀制备的组织块为样本，检测未固定与固定样本的基因表达变化相关性，得到决定系数R²=0.97，表明固定过程未引入显著的实验伪影。以相同方式制备的固定样本与新鲜样本的测序深度结果具有可比性（偏差±20%），且变异系数百分比（%CV）相近（分别为11.5%与12.7%），提示固定并未导致可检测RNA发生显著损失。无论组织切片是否经过固定，TempO-Seq™实验的灵敏度均保持一致。该实验在人、小鼠或大鼠的全转录组分析中表现均无差异。从5μm厚度切片上获取的10mm²与2mm²组织区域的分析结果一致，由此证明该实验具备高灵敏度，可对切片内的局灶组织学区域进行表达谱分析。不同组织区域的重复实验重现性因组织类型而异，决定系数R²范围为0.83（肺组织）至0.96（肝组织），9种组织类型的平均相关系数R²为0.90。对于计数大于200的基因，平均变异系数百分比为16.8%；计数大于50的基因则为20.3%。本研究鉴定出的组织特异性基因表达差异与已有文献报道相符。对存档时长可达30年的FFPE组织进行实验时，其性能未受到显著影响。同理，H&E染色对实验性能的影响可忽略不计，这有助于精准识别并刮取切片内特定组织学的小局灶区域，用于后续实验分析。