Supplementary Material for: Digital Multiplexed Gene Expression Analysis of mRNA and miRNA from Routinely Processed and Stained Cytological Smears: A Proof-of-Principle Study

Objective: Although transcriptomic assessments of small samples using high-throughput techniques are usually performed on fresh or frozen tissues, there is a growing demand for those performed on stained cellular specimens already used for diagnostic purposes. Study Design: The possibility of detecting mRNAs and microRNAs (miRNAs) from routinely processed cytological samples using nCounter® technology was explored. Fresh samples from pleural and peritoneal effusions were analyzed using 2 parallel methods: samples were smeared and routinely stained using the May-Grünwald-Giemsa or Diff-Quik® method and mounted using conventional methods, and they were also studied following a snap freezing method, in which samples were maintained at −80°C until use. mRNAs and miRNAs were assessed and compared after total RNA extraction from both routinely processed samples and their matched frozen controls. Results: A good concordance was found between the gene expression measured in routinely processed samples and their matched frozen controls for the majority of mRNAs and miRNAs tested. However, the standard deviation of low-expressed miRNA was high. Conclusions: Although nCounter® technology is a robust method to measure and characterize both mRNAs and miRNAs from routinely processed cytological samples, caution is recommended for the interpretation of low-expressed miRNA.

研究目标：尽管利用高通量技术对微量样本开展转录组分析通常以新鲜或冷冻组织为材料，但当前针对已用于临床诊断的染色细胞标本进行此类分析的需求正日益增长。
研究设计：本研究探讨了采用nCounter®技术从常规处理的细胞学样本中检测信使RNA（mRNAs）和微小RNA（microRNAs，简称miRNAs）的可行性。针对胸膜腔和腹膜腔积液的新鲜样本，采用两种平行方案进行分析：其一为将样本涂片后，通过迈-格-吉（May-Grünwald-Giemsa）染色法或Diff-Quik®染色法完成常规染色，并以常规方法封片；其二为采用速冻法处理样本，将其保存于-80℃直至实验使用。分别从常规处理样本及匹配的冷冻对照样本中提取总RNA，随后对mRNAs和miRNAs进行检测并对比分析。
研究结果：多数受检的mRNAs和miRNAs的基因表达量在常规处理样本与匹配的冷冻对照样本之间呈现出良好的一致性。但低表达miRNA的标准差较高。
研究结论：尽管nCounter®技术是一种可用于检测并表征常规处理细胞学样本中mRNAs和miRNAs的稳健方法，但在解读低表达miRNA的实验结果时需谨慎。