Background

Human tissue samples provide strong clinically relevant insight into disease processes that in vitro and in vivo systems cannot fully replicate. However, they pose some experimental challenges, such as heterogeneity and limited sample availability. Similarly, in vitro models, including cultured 3D models, possess their own limitations, as they do not recreate the integrated physiological environment of human disease. Animal models remain essential for mechanical and therapeutic studies, but translational issues often arise due to species-specific differences. For example, though human and murine macrophage subtypes share broad functional similarities, they diverge in important clinically relevant pathways, such as species-specific activation patterns of the PPARγ pathway [1]. Therefore, patient-derived tissue remains the most reliable source of directly translatable information.

The limited size and availability of patient samples restrict the number of downstream analyses that can be performed. This scarcity means that researchers often must be strategic about which assays to perform, given the limited number of samples available. To address this challenge, this protocol enables the tandem extraction of high-quality RNA and protein from the same tissue sample. This approach maximizes the usage of valuable human specimens while reducing intrasample variability, which may arise from sampling heterogeneous tissues.

This method is optimized for small tissue sections (15–30 mg), allowing more experimental conditions and technical replicates to be included. The use of RNAlater stabilizes RNA and protein, preserving integrity for one week at room temperature, one month at 4 °C, or indefinitely at -20 °C or lower [2]. Unlike common commercial methods like TRIzol, this workflow avoids toxic organic solvents like phenol and chloroform.

We developed and validated this protocol primarily with ex vivo–treated atherosclerotic plaque tissue from carotid endarterectomy (CEA). This tissue type provides substantial challenges because carotid plaques are often highly calcified due to long-term inflammation, necrosis, mechanical stress, aging, and osteogenic pathways, which are hallmarks of atherosclerotic disease. CEA tissue is also often limited in size and surgical availability. The use of small 15–30 mg (3 × 3 × 3 mm) sections for both RNA and protein could address some of these limitations. These small pieces of carotid tissue also remain metabolically active when cultured ex vivo for at least 24 h and are reactive to stimuli over this timeframe. This allows for the ex vivo treatment and screening of compounds of interest directly in human atherosclerotic tissue. The possibility of using small pieces of tissue and extracting both protein and RNA from them maximizes experimental outputs.

In addition to human CEA samples, we confirm the suitability of this protocol for use in mouse aortic tissue and THP-1 cells, and we expect this workflow to be adaptable to a wide range of tissue and sample types with minimal optimization (primarily in tissue volume and homogenization), because it relies on standard column-based RNA extraction kits and commonly available laboratory reagents. As a result, the protocol is widely accessible and compatible with most laboratory settings, providing an opportunity to maximize experimental readouts in most settings and a cheaper alternative to commercially available kits such as the Allprep (Qiagen) and Ambion (Thermo Fisher Scientific) kits. It also provides a layer of RNA stabilization, negating the need for liquid nitrogen snap freezing and tissue grinding for RNA extraction from tissue.

While several protocols have been described for precipitating total protein from various sources, including RNA extraction kit flowthrough [3–6], the protocol described here offers several distinct advantages. It avoids the use of hazardous organic compounds such as trichloroacetic acid, acetone, methanol, or chloroform. By avoiding these harsh organic compounds and avoiding washing with 96%–100% ethanol (as described in [6]), this protocol avoids stripping the proteins completely of hydration, facilitating easier downstream solubilization. Furthermore, by using only the first flowthrough of the RNA-extraction procedure for protein precipitation, samples can be processed in smaller volumes and tube sizes, reducing reagent and plasticware consumption.