Dissection

RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. In particular, Smart-3SEQ's compatibility with FFPE tissue unlocks an enormous number of archived clinical samples; combined with LCM it allows unprecedented studies of small cell populations and single cells isolated by their in situ context.

BackgroundFormalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA-protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult.MethodsWe excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step.ResultsWe successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well.ConclusionsWe present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.

BackgroundPulmonary sequestration (PS) is a form of congenital pulmonary malformation that is generally diagnosed in childhood or adolescence and usually resected when diagnosed. We aim to identify the clinical presentation and course of patients diagnosed to have PS during adulthood.MethodsUsing a computer-assisted search of Mayo clinic medical records, we identified adult patients with PS diagnosed between 1997 and 2016. Clinical and radiological data were collected including postoperative course for those who underwent surgical resection.ResultsWe identified 32 adult patients with PS; median age at diagnosis was 42 years (IQR 28-53); 17 patients (53%) were men. The median sequestration size was 6.6 cm (IQR 4.4-9.3). The type of sequestration was intralobar in 81% and extralobar in 19%. The most common location was left lower lobe posteromedially (56%). Forty-seven percent of the patients presented with no relevant symptoms. The most common radiographic finding was mass/consolidation in 61% and the most common feeding artery origin was the thoracic aorta (54%). Surgical resection was performed in 18 patients (56%) and postoperative complication was reported in 5 patients (28%). There was no surgical mortality. Median duration of follow-up after diagnosis for unresected cases, most of whom were asymptomatic, was 19 months (IQR 4-26) with no complications related to the PS reported.ConclusionsNearly one-half of adult patients with PS present with no relevant symptoms. The decision regarding surgical resection needs to weigh various factors including clinical manifestations related to PS, risk of surgical complications, comorbidities, and individual patient preferences.

Laser capture microdissection (LCM) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables spatial proteomics at the few-cell level but is constrained by cumulative losses during specimen capture, surface adsorption during processing, and sample transfer prior to LC-MS/MS analysis. The capture-associated losses are particularly relevant for pressure catapulting systems such as the legacy Zeiss PALM MicroBeam, which, despite discontinuation, remains in active use and therefore requires compatible low-loss workflows. We present MR-SP2 (microreactor-based sample preparation for spatial proteomics), a one-pot workflow integrating reproducible Zeiss LCM-cut specimen capture, processing with minimized adsorptive losses, and pipetting-free transfer with Evotip disposable precolumns. The workflow was evaluated using a formalin-fixed paraffin-embedded (FFPE) murine kidney tissue analyzed by timsTOF flex LC-MS/MS analysis. Across 50,000 μm3 regions (22 cells), MR-SP2 modestly improved proteome depth (3381 ± 80 versus 3174 ± 59 proteins). Decreasing sample input further accentuated the advantage of MR-SP2 in maintaining higher identification rates, highlighting the successful reduction of the adsorptive losses. At 12,500 μm3 (5-6 cells), identifications increased to 1145 ± 188 versus 302 ± 126. At 3125 μm3 (1-2 cells), identifications reached 695 ± 112 versus 206 ± 51. MR-SP2 improves identification depth for few-cell FFPE samples by nearly 3-fold compared to conventional tube-based workflows.