Impact of soft tissue homogenization methods on RNA quality - brief report

Sep 23, 2026

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High-quality RNA isolation from tissues underpins transcriptomic analysis, and tissue disruption methods strongly influence RNA integrity and purity. This brief report compares three homogenization approaches—mortar and pestle, ball mill, and a tissue homogenizer—across head and neck cancerous tissues, matched healthy free-margin tissues, and breast skin samples. RNA quantity and quality were evaluated via concentration/absorbance ratios, RIN, and reference gene Ct values. Results indicate the tissue homogenizer yields the highest purity and RIN, while Ct values show no significant method-dependent differences.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages7

Reading Time18 min

AccessFree to download

Abstract

Background

Materials and methods

Biological material

Mortar and pestle

Ball mill

Tissue homogenizer

Molecular Biology Reports (2025) 52:425 [https://doi.org/10.1007/s1](https://doi.org/10.1007/s1) 1033-025-10508-0 BRIEF REPORT Impact of soft tissue homogenization methods on RNA quality Julia Ostapowicz1,2,3 · Bartosz Maćkowiak1,4,5 · Kamila Ostrowska1,5 · Barbara Kaczmarek6 · Natalia Pietras6 · Dawid Frąckowiak7 · Magdalena Fundowicz8 · Katarzyna Kulcenty1 · Wojciech Golusiński5 · Wiktoria Suchorska1,2 Received: 13 January 2025 / Accepted: 14 April 2025 / Published online: 25 April 2025 © The Author(s) 2025 Abstract Background High-quality RNA isolation from tissues is crucial for transcriptomic analysis. The tissue disruption influences the RNA quality. The aim of this study was to compare different methods oftissue homogenization. Methods Three homogenization methods were used (mortar and pestle, ball mill, and tissue homogenizer) to disrupt head and neck cancerous tissues, healthy tissues from free margin of head and neck cancer patients, and breast skin from breast cancer patients. The comparison of isolated RNA quantity and quality by measuring the concentration and absorbance, RIN, and Ct values of reference genes were performed. Results RNA isolated after tissue homogenizer usage has the highest 260/230 ratio (p= 0.02) and concentration (p= 0.02) also RIN values tend to be highest across all studied tissues. There are no significant differences between Ct values across all tissues processed with different homogenization methods; however, the Ct values of GAPDHand S18 are negatively correlated with RIN number (p= 0.002, p= 0.003) . Conclusion The tissue homogenizer is the most suitable for obtaining high-quality RNA across all examined tissues, which is essential in various RNA-based analyses. GAPDHand S18 Ct can indicate the RNA quality measured by RIN values. Keywords Tissue homogenization · RNA isolation · Mortar and pestle · Ball mill · Tissue homogenizer 􀀍 Bartosz Maćkowiak[barmackowiak@gmail.com](barmackowiak@gmail.com) 1 Radiobiology Laboratory, The Greater Poland Cancer Centre, Poznan 61-866, Poland 2 Department of Electroradiology, Poznan University of Medical Sciences, Poznan 60-66, Poland 3 Doctoral School, Poznan University of Medical Sciences, Poznan 60-812, Poland 4 Doctoral School of Molecular Medicine, Medical University of Lodz, Lodz 90-700, Poland 5 Department of Head and Neck Surgery, Poznan University of Medical Sciences, The Greater Poland Cancer Centre, Poznan 61-866, Poland 6 Faculty of Chemistry, Adam Mickiewicz University Poznan, Poznan 61-614, Poland 7 Center for Advanced Technologies, Adam Mickiewicz University Poznan, Poznan 61-614, Poland 8 Radiotherapy Ward I, Greater Poland Cancer Centre, Poznan, Poland Background Extracting high-quality genetic material from human tissue is crucial for various molecular applications, including standard laboratory procedures and high-throughput analysis [1, 2] . Proper methods oftissue homogenization must allow for the recovery of as much genetic material as possible with simultaneous preservation. The clinical samples are limited in size, are one-time collected; thus, selecting a established homogenization method is essential. Tissues delivered from different solid tumors, including head and neck cancers (HNC), are challenging in molecular processing caused by histologic and genetic heterogeneity. Also, skin needs a specific approach due to the presence of hyaluronic acid, collagen fibers, and surface nucleases [3] . There are several methods of tissue homogenization, including mechanical disruption or enzymatic digestion [4, 5] . Previous works emphasize challenges of patients’heterogeneous tissue homogenization. Ivanov et al. used mortar and pestle to ground very hard tissue such as cartilage, and isolated RNA presents satisfaction RIN values [6] . Usage of tissue homogenizator is common and various protocols are publicized [7] . Another method that could be used for tissue homogenization includes cryot

FAQs

Which homogenization method produced the highest RNA purity and RIN values?

RNA isolated after using the tissue homogenizer showed the highest 260/230 ratio and the highest RIN values tendency across examined tissues.

Were Ct values different across tissues processed with different homogenization methods?

No significant differences in Ct values were found across tissues processed with the different homogenization methods.

How did GAPDH and S18 Ct relate to RNA quality?

GAPDH and S18 Ct values were negatively correlated with RIN values, indicating they can reflect RNA quality.

Sep 23, 2026

Upload by James