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Prospects of transcript profiling for mRNAs and MicroRNAs using formalin-fixed and paraffin-embedded dissected autoptic multiple sclerosis lesions.

ABSTRACT: The elaboration of novel pathogenic aspects of multiple sclerosis (MS) requires the analysis of well-defined stages of lesion development. However, specimens of certain stages and lesion types are either present in small brain biopsies, insufficient in size for further molecular studies or available as formalin-fixed and paraffin-embedded (FFPE) material only. Therefore, application of current molecular biology techniques to FFPE tissue is warranted. We compared FFPE and frozen tissue by using quantitative polymerase chain reaction and report: (1) FFPE material is highly heterogeneous regarding the utility for transcript profiling of mRNAs; well-preserved FFPE samples had about a 100-fold reduced sensitivity compared with frozen tissue, but gave similar results for genes of sufficient abundance; (2) FFPE samples not suitable for mRNA analysis are still highly valuable for miRNA quantification; (3) the length of tissue fixation greatly affects utility for mRNA but not for miRNA analysis; (4) FFPE samples can be processed via a hot water bath for dissection of defined lesion areas; and (5) in situ hybridization for proteolipid protein (PLP) helps to identify samples not suitable for mRNA amplification. In summary, we present a detailed protocol how to use autoptic FFPE tissue for transcript profiling in dissected tissue areas.

SUBMITTER: Eisele S

PROVIDER: S-EPMC8057623 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:BackgroundRheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovial joints. Early intervention followed by early diagnosis can result in disease remission; however, both early stage diagnosis and provision of effective treatment have been impeded by the heterogeneity of RA, which details of pathological mechanism are unclear. Regardless of numerous investigations of RA by means of genomic and proteomic approaches, proteins interplaying in RA synovial tissues that contain various types of synoviocytes, are not yet sufficiently understood. Hence we have conducted an HPLC/mass spectrometry-based exploratory proteomic analysis focusing on synoviocyte lesions laser-microdissected (LMD) from formalin-fixed paraffin-embedded (FFPE) synovial tissues (RA, n = 15; OA, n = 5), where those of Osteoarthritis (OA) were used as the control.ResultsA total of 508 proteins were identified from the RA and OA groups. With the semi-quantitative comparisons, the spectral index (SpI), log2 protein ratio (R SC ) based on spectral counting, and statistical G-test, 98 proteins were found to be significant (pair-wise p < 0.05) to the RA synovial tissues. These include stromelysin-1 (MMP3), proteins S100-A8 and S100-A9, plastin-2, galectin-3, calreticulin, cathepsin Z, HLA-A, HLA-DRB1, ferritin, neutrophil defensin 1, CD14, MMP9 etc.ConclusionsOur results confirmed the involvement of known RA biomarkers such as stromelysin-1 (MMP3) and proteins S100-A8 and S100-A9, and also that of leukocyte antigens such as HLA-DRB1. Network analyses of protein-protein interaction for those proteins significant to RA revealed a dominant participation of ribosome pathway (p = 5.91 × 10(-45)), and, interestingly, the associations of the p53 signaling (p = 2.34 × 10(-5)). An involvement of proteins including CD14, S100-A8/S100-A9 seems to suggest an activation of the NF-kB/MAPK signaling pathway. Our strategy of laser-microdissected FFPE-tissue proteomic analysis in Rheumatoid Arthritis thus demonstrated its technical feasibility in profiling proteins expressed in synovial tissues, which may play important roles in the RA pathogenesis.

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Prospects of transcript profiling for mRNAs and MicroRNAs using formalin-fixed and paraffin-embedded dissected autoptic multiple sclerosis lesions.

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