CryoGrid-PIXUL-RNA: High throughput RNA isolation platform for tissue transcript analysis

Abstract

Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in life sciences analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such specimens remain, in comparison, inefficient. To mitigate this bottleneck, we have developed a low-cost user-friendly system, the CryoGrid, that consists of CryoBlock, thermometer/thermocouple, and QR coded CryoTrays to freeze and store frozen tissue fragments, and hand-held CryoCore tool for tissue sampling supported by iPad and Google apps to display tissues, direct coring and share metadata. RNA is one of the most studied analytes. There is a decades-long history of developing methods to isolate and analyze RNA. Still, the throughput of sampling and RNA extraction from tissues has not matched that of the high throughput transcriptome analytical platforms. To address this need, we have integrated the CryoGrid system with PIXUL-based methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. TRIzol is commonly used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. We developed a PIXUL-based TRIzol-free RNA isolation fast protocol that uses a buffer containing proteinase K (PK). Virtually every disease (and often therapeutic agents' toxicity) is a systemic syndrome but often only one organ is examined. CryoGrid-PIXUL, integrated with either TRIzol or PK buffer RNA isolation protocols, yielded similar RNA profiles in a multiorgan (brain, heart, kidney and liver) mouse model of sepsis. Thus, RNA isolation using the CryoGrid-PIXUL system combined with the PK buffer offers an inexpensive user-friendly workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

MultiomicsTracks96: A high throughput PIXUL-Matrix-based toolbox to profile frozen and FFPE tissues multiomes

“…PIXUL offers unparalleled sample preparation throughput to explore multiple samples in one experiment (27), but the upstream steps such as cryostorage and particularly tissue sampling represented a challenge in the workflow of multi-omics analysis. To mitigate this bottleneck, we designed a high-throughput platform for storing frozen tissues and sampling, CryoGrid, which includes the CryoCore (34). CryoCore is a hand-held rotary tool for rapid sampling of frozen tissues for molecular analysis and histology.…”

“…CryoCore is a hand-held rotary tool for rapid sampling of frozen tissues for molecular analysis and histology. With CryoCore, the same frozen tissue can be sampled multiple times, yielding reproducible core sizes (34). We used mouse brain, heart, kidney, liver, lung, and muscle fragments to integrate and optimize CryoGrid and CryoCore with PIXUL protocol to extract RNA and chromatin from frozen tissue samples ( Fig.1A ).…”

“…Almost any disease, and often therapeutic intervention, are systemic conditions where animal models provide the means to understanding multiorgan dysfunction (33). To store and sample large numbers of biospecimens for multi-omics analysis, we designed a platform for cryostoring multiple tissue samples and engineered a hand-held rotary tool for rapid sampling of frozen tissues, CryoGrid system, which consists of CryoBox, CryoBlock, thermometer/thermocouple, QR barcoded CryoTrays, and CryoCore (34).…”

“…Frozen tissues were placed in the wells and immobilized with either OCT or Leica CryoGel. The CryoTray, with tissues covered by a QR code labeled lid, was stored at -80 o C (34).…”

Analysis of gliomas DNA methylation: Assessment of pre-analytical variables

“…Frozen tissues are placed in the wells and immobilized with either OCT or Leica CryoGel. The CryoTray, with tissues covered by a QR code labeled lid, is stored at -80 o C (23). Tissues are sampled with CryoCore and cores are jetted into tubes or 96-well PIXUL plates.…”