Breaking the seals: Efficient mRNA detection from human archival paraffin-embedded tissue

Abstract

During our study on HOXA13, HOXD12, and HOXD13 mRNA expression in human adult and embryonic tissues, we were confronted with the fact that, within our specimen collection, as in other University Departments in Europe, <20% of all samples yielded reliable labeling, while most samples were resistant to hybridization by standard protocols due to over-fixation. Fixation is essential for specimen stability, especially when samples are stored at room temperature and used for histology, and people tend to be more worried about under-than over-fixation. On the other hand fixation inhibits penetration by the probe and may also trap mRNA within ribosomes. Therefore, we developed a nonradioactive in situ hybridization technique, which allows detection of mRNA expressed on low levels from a variety of differentially fixed tissues while maintaining tissue integrity. This was achieved by improving target retrieval and probe detection. In contrast with others, our method allows reliable staining from tissues that are fixed in paraformaldehyde from four hours to over one week, and archived samples that were stored at room temperature for several years (17-19 yr in some cases) and exceeds detection limits of purely fluorescent methods. Our protocol is highly suitable for detecting CDX-2 mRNA in carcinoma specimens, but especially designed to investigate mRNAs in nonpathological adult and embryonic tissues. Due to the use of standardized probes, we do not expect problems in detecting other mRNAs expressed in suitable amounts.

Back to the start: Evaluation of prognostic markers in gastrointestinal stromal tumors

“…Furthermore, we were unable to confirm the role of differentiation and cell-cycle regulatory proteins, such as Bcl-2, P16 and P53, as well as CD44, as prognostic factors, similar to previous studies (8,(21)(22)(23)(24). The reasons may be different antibodies, antigen retrieval or inter-observer variations of the immunohistochemical staining (10,11,15).…”

“…The general characteristics of the investigated GISTs are summarized in Table I. The range of fixation time for the obtained specimens was determined between 12 and 24 h in 4% phosphate-buffered saline-formalin solution to avoid false-positive or -negative immunohistochemical staining patterns due to under-or over-fixation (10,11).…”

Spatio‐temporal expression of HOX genes in human hindgut development

“…7B). It is noteworthy that the expression of HOX genes was suppressed in close vicinity to rectal cancer samples (Illig et al, 2009), thereby outlining the difference between CDX2‐positive tumor from healthy HOX ‐gene‐positive tissue by a thin layer of cells that were negative for both HOX and CDX2. In contrast, other studies in human rectal tissues reported the lack of HOXD13 expression in normal tissue but an increase in neoplastic rectal tissue (Cantile et al, 2009).…”

“…For a better understanding of the processes involved in the development of the human hindgut, we collected specimens covering the age of developmental week 6 to 12. We recently developed a reliable method to investigate the expression of mRNAs in newly collected as well as archival samples (Illig et al, 2009, 2010). Applying this method, we present data demonstrating a clearly restricted temporal and spatial expression pattern of nine HOX gene members of the posterior clusters in human hindgut development and in adulthood.…”

Robust linear regression model of Ki-67 for mitotic rate in gastrointestinal stromal tumors

“…The range of fixation time of the obtained specimens was between 12 and 24 h in 4% phosphate-buffered saline solution to avoid false-positive or -negative immunohistochemical staining patterns due to under- or over-fixation (14,15). …”