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

Abstract

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.

Long-Term Stability of Human Genomic and Human Papillomavirus DNA Stored in BD SurePath and Hologic PreservCyt Liquid-Based Cytology Media

“…Prior reports of variable success in RNA and DNA purification from BD SurePath-and Hologic PreservCyt-preserved specimens over shorter storage periods may also be explained by the ability of the extraction method to reverse fixative-induced cross-linking or other deleterious effects on the cells and their nucleic acid that render it refractory to lysis and extraction. Extraction methods that utilize high temperature and/or proteinase K digestion for extended periods reported higher success rates in DNA detection and the ability to detect longer fragments (5,16). This suggests that prior reports of nucleic acid degradation following storage in LBC media need to be reevaluated and may have been limited by the extraction and amplification methods used rather than degradation of the target nucleic acid.…”

“…However, we consider this unlikely given that the samples in this study also experienced room temperature storage and almost half of them had aged beyond their recommended expiration date prior to initial refrigerated storage and testing in our laboratory. FFPE specimens are also routinely stored at room temperature for extended periods, and nucleic acids can be successfully isolated from these specimens using optimized extraction procedures (16,26).…”

“…Formalin treatment results in reversible cross-linking of proteins to DNA, which can render it refractory to magnetic-or silica-based purification and subsequent downstream nucleic acid biochemistry, such as PCR (13). Cross-linking can be removed using proteinase K digestion and/or heat (14)(15)(16).…”

Section I: Integrating laboratory medicine with tissue specimens

“…9 Although high-quality RNA is consistently extracted from fresh tissue and therefore is preferred for some applications, 10 methods can be optimized to achieve successful RNA extraction for nearly all fixed tissue specimens, including FFPE tissue blocks. 11,12 Many surgical cases, and neurosurgical cases in particular, generate requests for intraoperative consultations (frozen sections), and this tendency can be used for appropriately allotting tissue for future studies. Not only can the pathologist render a preliminary differential diagnosis via intraoperative smear or frozen section analysis or both, but he or she can also make a judgment as to the composition and viability of the lesion tissue.…”

Technique and method development for intervertebral disc (IVD) research

“…There were also a number of studies by other groups using Taqman based gene expression analysis with RNA extracted with TRIzol reagent [156][157][158][159][160][161][162] …”