Abstract
Human tissue samples represent the gold standard for obtaining clinically relevant and translatable insight into disease processes that in vitro systems cannot fully reproduce. However, patient-derived samples are often limited in size and availability, limiting the number of downstream assays that can be performed. To maximize the use of invaluable human samples, we present a protocol for the tandem extraction of high-quality RNA and protein from the same tissue section. This method has been optimized for 15-30 mg tissue sections, enabling more experimental conditions and technical replicates, while minimizing intrasample variability associated with heterogeneous tissues. This protocol also avoids potentially hazardous solvents present in phenol-chloroform-based methods such as TRIzol, providing a safer and more accessible workflow without compromising biomolecule integrity. This protocol was developed and validated using atherosclerotic plaque tissue from carotid endarterectomy, a very challenging tissue type to work with due to extensive calcification, necrosis, and limited surgical availability. We have also validated this method using mouse aortic tissue and cultured THP-1 cells, demonstrating its versatility across sample input types. As this protocol relies on standard column-based RNA extraction kits and commonly available reagents for protein precipitation and extraction, this methodology is widely accessible and easy to implement as a standard, streamlined workflow. Key features • Advances ex vivo models of human disease (e.g., atherosclerosis) by maximizing the use of patient-derived tissue samples. • Minimizes patient variability by generating directly comparable RNA and protein readouts. • Minimizes the number of patient samples required and maximizes the output from each sample. • Stabilizes RNA integrity from tissue without impacting the quality of proteins derived from the same sample.