Storage Conditions and Immunoreactivity of Breast Cancer Subtyping Markers in Tissue Microarray Sections.

Loss of immunoreactivity in tissue sections has been shown to occur when slide sections are stored at room temperature for prolonged periods of time. We conducted a systematic investigation to determine the extent of staining loss in various storage conditions to determine an optimal storage method. We investigated 6 antibodies that are commonly used for breast cancer subtyping in research studies with immunohistochemistry (ER, PR, HER2, CK5/6, EGFR, and Ki67) in formalin-fixed paraffin-embedded breast tissue microarrays consisting of 148 patients. Tissue microarrays were sectioned at various time points: fresh, 1 week, 1 month, 6 months, and 12 months before staining. Slides sectioned at each time point were stored in 5 storage conditions: desiccator, paraffin dipped, 4°C, -20°C, and -80°C. Immunohistochemistry scores were assessed over time with McNemar Test and Bowker Test of Symmetry. Desiccator storage was the only storage condition that did not show any loss in immunoreactivity for any antibody or time point in our study. Paraffin coated slides were the most difficult storage method operationally and also showed the most loss in immunoreactivity. Storing sections in a desiccator was the most effective method for minimizing immunoreactivity loss. Cold storage at 4°C is an intermediate option that is not as protective as a desiccator, but offers the advantage of being accessible to virtually all research labs.

Maximizing RNA yield from archival renal tumors and optimizing gene expression analysis.

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen's TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers' protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan qPCR can be optimized by using the MasterPure RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.