Efficient mRNA detection from human archival paraffin-embedded tissue: An update

Abstract

We recently published an in situ hybridization protocol for archival tissue using a commercial hybridization buffer. This buffer is no more available. Therefore, we have developed an improved protocol with a defined hybridization buffer.

We recently published a method describing the technical background for mRNA studies on tissues processed for immunohistochemistry or histology (Illig et al. 2009). This novel protocol enables the investigation of archival tissues collected over at least the last two decades, not only involving developmental studies, but also for virtually all questions related to gene expression. In this protocol, we suggested the use of HybriBuffer ISH, supplied by Biognostik. This company no longer exists, and we have been unable to find another supplier. Therefore, we have developed a hybridization buffer that yields essentially the same results using the published protocol. The buffer is based on 4× saline sodium citrate (SSC) and 50% formamide. This is supplemented with sodium phosphate buffer (50 mM at pH 6.5), Denhard solution (0.1×), tRNA (0.25 mg/mL), single-stranded salmon testes DNA (2.5 mg/mL), EDTA (1 mM at pH 8.0), and sodium dodecyl sulfate (SDS) (0.04%). The concentrations in parentheses are calculated with respect to the final volume including formamide. The 50× Denhard solution was made from 1% Ficoll 400, 1% polyvinylpyrrolidone MW 360.000, and 1% bovine serum albumin, and was filtrated (0.45 μm). Single-strand DNA was denatured for 10 min at 95°C before adding it to the buffer. The pH was adjusted to 7.4. Besides the SSC and formamide, the concentration of SDS is crucial. Since SDS might damage the subsequently used antibody, we recommend changing the stringent washing buffer once to avoid contamination of the antibody solution with SDS.

Replacing HybriBuffer ISH with this buffer renders the differential addition of Triton X-100 to fixed tissues unnecessary. In the current protocol, 0.4% Triton X-100 is suitable for all kinds of samples investigated. We tested the replacement buffer with samples obtained from adult and embryonic tissue, fixed and stored for different times, and did not detect any differences compared with the previously published results (Fig. 1).

FIGURE 1.

Photomicrographs of adult (a,c) and fetal (gynecological age ∼12 wk) (b,d) tissue samples are depicted after in situ hybridization for HOXA13 mRNA. Photomicrographs of adult tissue depict the muscular layer of the proximal rectum. For fetal samples, epithelial and mesenchymal cells of the hindgut are displayed. (Upper panels) Sections processed with the new hybridization buffer; (lower panels) sections processed with HybriBuffer ISH. Note the improved conservation of tissue integrity with the new buffer. Scale bars in c (for a–d) and in the inlay of c (for inlays in a–d), represent 100 and 10 μm, respectively.