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Updated: Jul 5, 2026
MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues
Published on: November 30, 2013
Canine urothelial carcinoma: a pilot study of microRNA detection in formalin-fixed, paraffin-embedded tissue samples
Mara S Varvil1, Samuel L Clark1, Taylor W Bailey1
1Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA.
Abstract:
We assessed the effects of fixation time in formalin and inclusion of surrounding tissue on microRNA (miRNA) cycle quantification (Cq) values in formalin-fixed, paraffin-embedded (FFPE) urothelial carcinoma (UC) tissue (n = 3), and the effect of conditions on miRNAs in urine from 1 healthy dog. MiRNAs were extracted using commercial kits and quantified using miRNA-specific fluorometry in normal bladder tissue scrolls, UC tissue cores, and bladder muscularis tissue cores from 4 FFPE bladder sections (3 UCs, 1 normal), plus 1 UC stored in formalin for 1, 8, 15, and 22 d before paraffin-embedding. Urine was collected from a healthy dog on 4 occasions; 1-mL aliquots were stored at 20, 4, -20, and -80°C for 4, 8, 24, and 48 h, and 1 and 2 wk. For both FFPE tissue and urine, we used reverse-transcription quantitative real-time PCR (RT-qPCR) to quantify miR-143, miR-152, miR-181a, miR-214, miR-1842, and RNU6B in each tissue or sample, using miR-39 as an exogenous control gene. The Cq values were compared with ANOVA and t-tests. The time of tissue-fixation in formalin did not alter miRNA Cq values; inclusion of the muscularis layer resulted in a statistically different miRNA Cq profile for miR-152, miR-181a, and RNU6B in bladder tissue. MiRNAs in acellular urine were stable for up to 2 wk regardless of the storage temperature. Our findings support using stored FFPE and urine samples for miRNA detection; we recommend measuring miRNA only in the tissue of interest in FFPE sections.
Insights
Formalin fixation time did not impact microRNA (miRNA) quantification in urothelial carcinoma tissue. However, including bladder muscularis tissue altered miRNA profiles. Urine miRNAs remained stable for two weeks, supporting the use of stored samples for miRNA detection.
Area of Science:
- Veterinary Pathology
- Molecular Biology
- Biochemistry
Background:
- MicroRNAs (miRNAs) are crucial biomarkers in urothelial carcinoma (UC).
- Formalin-fixed, paraffin-embedded (FFPE) tissues and urine samples are commonly used for miRNA analysis.
- Standardization of pre-analytical conditions is essential for reliable miRNA quantification.
Purpose of the Study:
- To evaluate the impact of formalin fixation time and tissue inclusion on miRNA quantification in FFPE UC samples.
- To assess miRNA stability in urine under various storage conditions.
- To provide recommendations for miRNA analysis in FFPE tissues and urine.
Main Methods:
- MiRNAs were extracted from FFPE urothelial carcinoma and normal bladder tissues, including muscularis.
- Urine samples were collected and stored at different temperatures and durations.
- Reverse-transcription quantitative real-time PCR (RT-qPCR) was used to quantify specific miRNAs (miR-143, miR-152, miR-181a, miR-214, miR-1842, RNU6B) with miR-39 as a control.
Main Results:
- Formalin fixation time did not significantly alter miRNA cycle quantification (Cq) values.
- Inclusion of the muscularis layer in FFPE bladder tissue resulted in statistically different miRNA Cq profiles for miR-152, miR-181a, and RNU6B.
- MiRNAs in acellular urine remained stable for up to two weeks, irrespective of storage temperature.
Conclusions:
- Stored FFPE tissues and urine samples are suitable for miRNA detection.
- It is recommended to measure miRNAs specifically within the tissue of interest in FFPE sections to avoid confounding results.
- Standardized protocols for sample handling are crucial for accurate miRNA biomarker studies.
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