Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

BackgroundRetrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal FindingsFor effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. SignificanceWe determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies.背景：具有明确临床随访信息的存档人类标本回顾性研究，常用于识别疾病的预测性与预后性分子标志物。然而，由于生化特性差异，福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）样本的DNA与RNA通常需分别提取：要么从不同的组织切片提取，要么将蛋白酶K（proteinase-K）消化后的组织拆分后从同一切片提取。前者会阻碍准确的相关性分析，而后者在使用稀有或存量有限的存档标本时并不具备可行性。 主要研究结果：为了在不拆分蛋白酶K消化液的前提下，从单份FFPE标本中有效回收基因组DNA与总RNA，我们优化了一种基于TRIzol的共提取方法：分别从下层水相纯化DNA、上层有机相纯化RNA。我们使用7份不同的存档标本，评估了基于TRIzol的共提取方法回收的基因组DNA与总RNA总量，并将结果与两款商用试剂盒进行对比：一款是Qiagen AllPrep DNA/RNA FFPE试剂盒（用于共提取），另一款是Ambion RecoverAll™ 总核酸分离试剂盒（用于分别提取FFPE来源的DNA与RNA）。随后，为准确评估从单份FFPE标本中共提取的DNA与RNA的质量，我们采用实时定量逆转录PCR（qRT-PCR）、基因表达谱分析及甲基化检测技术，分析了配对的新鲜与FFPE处理的MCF10A细胞中回收的微小RNA（microRNAs）、信使RNA（mRNAs）及基因组DNA。实验结果表明，相较于另外两款试剂盒，基于TRIzol的共提取方法可回收更多的FFPE来源DNA与RNA，尤其能获得更高质量的微小RNA与基因组DNA，可用于后续的分子分析。 研究意义：我们证实，从单份FFPE标本中共提取基因组DNA与总RNA，是一种有效的回收策略，可获取高质量的实验材料，用于并行开展分子与高通量分析。我们优化的方法能够回收原本可能被丢弃或降解的DNA，用于后续的额外研究。