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Microwave pretreatment improves RNA-ISH in various formalin-fixed tissues using a uniform protocol
L Wilkens1, R von Wasielewski, M Werner
1Pathologisches Institut der Medizinischen Hochschule Hannover, Germany.
Abstract:
RNA is situ hybridization technique (RNA-ISH) is hampered by formalin fixation of tissues. This necessitates a pretreatment step of enzymatic digestion. However, to achieve the best results, digestion times and concentrations of enzymes have to be adapted for every specimen in a cumbersome procedure. Microwave pretreatment (MP) of formalin-fixed tissues has developed to become a powerful tool in immunohistochemistry (IHC) in recent years. To evaluate whether MP could also be helpful for RNA-ISH, we compared MP and conventional enzymatic pretreatment systematically using different tissues and various fixation times. As a model for RNA-ISH we chose the detection of kappa/lambda light chain mRNA in tonsils and lymph nodes with follicular hyperplasia and lymph nodes of patients with lymphocyte predominant Hodgkin's Disease (LPHD). Signal intensity obtained after MP was at least as good or dramatically enhanced as that obtained with optimized, single case adapted conventional pretreatment, the morphology being much better preserved after MP. These results confirm MP as a suitable method to unify the staining protocols in RNA-ISH, regardless of the duration of formalin fixation. Based on our results we recommend MP as a reliable and inexpensive method to enhance, standardized and simplify RNA-ISH.
Insights
Microwave pretreatment (MP) simplifies RNA in situ hybridization (RNA-ISH) by eliminating cumbersome enzymatic digestion. This method enhances RNA-ISH signals and preserves tissue morphology, offering a standardized approach for formalin-fixed tissues.
Area of Science:
- Molecular Biology
- Histology
- Biotechnology
Background:
- Formalin fixation of tissues significantly hinders RNA in situ hybridization (RNA-ISH) techniques.
- Conventional pretreatment for RNA-ISH often requires time-consuming enzymatic digestion, with protocols needing case-by-case optimization.
- Microwave pretreatment (MP) has emerged as an effective method for improving immunohistochemistry (IHC) on formalin-fixed tissues.
Purpose of the Study:
- To evaluate the efficacy of microwave pretreatment (MP) as an alternative to enzymatic digestion for RNA-ISH.
- To compare MP with conventional enzymatic pretreatment in terms of signal intensity and tissue morphology preservation.
- To assess the applicability of MP across various tissues and fixation durations for RNA-ISH standardization.
Main Methods:
- Systematic comparison of MP versus conventional enzymatic pretreatment for RNA-ISH.
- Utilized different tissue types, including tonsils and lymph nodes with varying pathological conditions (follicular hyperplasia, lymphocyte predominant Hodgkin's Disease).
- Focused on detecting kappa/lambda light chain mRNA as a model for RNA-ISH analysis.
Main Results:
- Microwave pretreatment (MP) yielded signal intensities comparable to or significantly enhanced compared to optimized enzymatic digestion.
- Tissue morphology was substantially better preserved following MP than with conventional enzymatic pretreatment.
- MP demonstrated effectiveness across diverse tissues and fixation times, indicating broad applicability.
Conclusions:
- Microwave pretreatment (MP) is a highly effective method for enhancing RNA-ISH signal and preserving tissue morphology.
- MP offers a standardized, simplified, and reliable protocol for RNA-ISH, irrespective of formalin fixation duration.
- Recommends MP as a valuable, inexpensive technique to improve and unify RNA-ISH staining procedures.