Molecular and Histological Evaluation of Sheep Ovarian Tissue Subjected to Lyophilization

Cryopreservation is routinely used to preserve cells and tissues; however, long time storage brings many inconveniences including the use of liquid nitrogen. Freeze-drying could enable higher shelf-life stability at ambient temperatures and facilitate transport and storage. Currently, the possibility to freeze-dry reproductive tissues maintaining vitality and functions is still under optimization. Here, we lyophilized sheep ovarian tissue with a novel device named Darya and a new vitrification and drying protocol and assessed effects on tissue integrity and gene expression. The evaluation was performed immediately after lyophilization (Lio), after rehydration (LR0h) or after two hours of in vitro culture (IVC; LR2h). The tissue survived lyophilization procedures and maintained its general structure, including intact follicles at different stages of development, however morphological and cytoplasmic modifications were noticed. Lyophilization, rehydration and further IVC increasingly affected RNA integrity and caused progressive morphological alterations. Nevertheless, analysis of a panel of eight genes showed tissue survival and reaction to the different procedures by regulation of specific gene expression. Results show that sheep ovarian tissue can tolerate the applied vitrification and drying protocol and constitute a valid basis for further improvements of the procedures, with the ultimate goal of optimizing tissue viability after rehydration.

Effect of RNA preservation methods on RNA quantity and quality of field collected avian whole blood

A limitation of comparative transcriptomic studies of wild avian populations continues to be sample acquisition and preservation to achieve resulting high-quality RNA (i.e., ribonucleic acids that transfers, translates, and regulates the genetic code from DNA into proteins). Field sampling of wild bird samples provides challenges as RNA degradation progresses quickly and because cryopreservation is often not feasible at remote locations. We collected blood samples from songbirds, as avian blood is nucleated and provides sufficient transcriptionally active material in a small and non-lethal sample, to compare the efficacy of widely available RNA stabilizing buffers, RNAlater (Ambion) and DNA/RNA Shield (Zymo) at differing concentrations along with a dry ice-based flash freezing method (Isopropanol 99% and dry ice mixture, -109°C). Each blood sample was divided among five different preservation treatments (dry ice-based flash freezing, RNAlater with 1:5 or 1:10 dilution, or DNA/RNA Shield with 1:2 or 1:3 dilution). A new protocol was optimized for total RNA extraction from avian blood samples with small starting volumes enabling sampling of small passerines. We quantified quality measures, RNA integrity numbers (RINe), rRNA ratios, and total RNA concentrations. We found that RNA preservation buffers, RNAlater and DNA/RNA Shield at all concentrations, provide sample protection from RNA degradation. We suggest caution against using dry ice-based flash-freezing alone for samples preservation as these samples resulted in lower quality measures then samples in preservation buffer. Total RNA concentration was generally not affected by preservation treatment and may vary due to differences in initial samples volumes and carryover across processing steps.

RNA Quality Control Non-Denaturing Agarose "Bleach" Gel v1

To evaluate the quality of RNA without the use of toxic chemicals, samples are run in a non-denaturing agarose "bleach" gel and product segregation is used to estimate RNA integrity. Be aware that in a non-denaturing gel, the RNA will not segregate strictly on particle size due to secondary structures of the molecules. This method is for an estimation of quality only and the location of the banding in relation to a base pair ladder does not allow confident determination of the size of the RNA fragment. If true fragment size determinations are required, RNA quality should be evaluated on a denaturing gel electrophoresis system.

Isolation of RNA from Acute Ischemic Stroke Clots Retrieved by Mechanical Thrombectomy

Mechanical thrombectomy (MT) for large vessel acute ischemic stroke (AIS) has enabled biologic analyses of resected clots. While clot histology has been well-studied, little is known about gene expression within the tissue, which could shed light on stroke pathophysiology. In this methodological study, we develop a pipeline for obtaining useful RNA from AIS clots. A total of 73 clot samples retrieved by MT were collected and stored in RNALater and in 10% phosphate-buffered formalin. RNA was extracted from all samples using a modified Chemagen magnetic bead extraction protocol on the PerkinElmer Chemagic 360. RNA was interrogated by UV–Vis absorption and electrophoretic quality control analysis. All samples with sufficient volume underwent traditional qPCR analysis and samples with sufficient RNA quality were subjected to next-generation RNA sequencing on the Illumina NovaSeq platform. Whole blood RNA samples from three patients were used as controls, and H&E-stained histological sections of the clots were used to assess clot cellular makeup. Isolated mRNA was eluted into a volume of 140 µL and had a concentration ranging from 0.01 ng/µL to 46 ng/µL. Most mRNA samples were partially degraded, with RNA integrity numbers ranging from 0 to 9.5. The majority of samples (71/73) underwent qPCR analysis, which showed linear relationships between the expression of three housekeeping genes (GAPDH, GPI, and HPRT1) across all samples. Of these, 48 samples were used for RNA sequencing, which had moderate quality based on MultiQC evaluation (on average, ~35 M reads were sequenced). Analysis of clot histology showed that more acellular samples yielded RNA of lower quantity and quality. We obtained useful mRNA from AIS clot samples stored in RNALater. qPCR analysis could be performed in almost all cases, while sequencing data could only be performed in approximately two-thirds of the samples. Acellular clots tended to have lower RNA quantity and quality.

The Department of Veterans Affairs Gulf War Veterans’ Illnesses Biorepository: Supporting Research on Gulf War Veterans’ Illnesses

Aims: To introduce resource supporting research on Gulf War illness (GWI) and related disorders, the Gulf War Veterans’ Illnesses Biorepository (GWVIB). Methods: Gulf War era veterans (GWVs) are recruited nationally and enrolled via telephone and email/postal mail. Enrolled veterans receive annual telephone and mail follow-up to collect health data until their passing. A postmortem neuropathological examination is performed, and fixed and frozen brain and spinal cord samples are banked to support research. Investigators studying GWI and related disorders may request tissue and data from the GWVIB. Results: As of September 2021, 127 GWVs from 39 states were enrolled; 60 met the criteria for GWI, and 14 met the criteria for chronic multisymptom illness (CMI). Enrollees have been followed up to six years. Postmortem tissue recoveries were performed on 14 GWVs. The most commonly found neuropathologies included amyotrophic lateral sclerosis, chronic traumatic encephalopathy, and Lewy body disease. Tissue was of good quality with an average RNA integrity number of 5.8 (SD = 1.0) and ≥4.8 in all of the cases. Discussion: The availability of health data and high-quality CNS tissue from this well-characterized GWV cohort will support research on GWI and related disorders affecting GWVs. Enrollment is ongoing.

PSXII-4 Impact of delayed processing time on total RNA quality and quantitation of transcript abundance

Critical biological information related to economically important traits is present in the blood transcriptome in cattle. Following a sample collection, however, RNA molecules containing this valuable information are prone to degradation affecting transcript abundance. Thus, proper sample handling and storage is essential when working with RNA. Here, we hypothesized that delayed time between sample collection and processing would lead to RNA degradation and alter gene transcript quantification. We aimed to determine the effect of delayed processing of whole blood on transcriptomic profiles in peripheral white blood cells (PWBC). We collected blood samples (10ml) in tubes containing anticoagulant (K2EDTA) from estrus synchronized beef heifers (n = 5). The tubes remained chilled on ice until processing time. From each heifer, we collected five samples from the jugular vein. We processed one sample from each animal within one hour of collection, and we delayed the processing time of the remaining samples to three, six, eight, and 24 hours post collection. We extracted total RNA from PWBCs, measured yield and assessed quality. We also quantified transcript abundance of 12,724 genes by RNA-sequencing. Samples processed 24 hours post collection had lower RNA integrity number (RIN) compared to samples processed withing one hour of collection (RIN24h=8.00±0.37, RIN1h=8.52±0.37, P = 0.03). There were four and 504 genes with differential transcript abundance (FDR< 0.05) when comparing eight and 24 hours of delayed processing with samples processed within one hour of collection, respectively. Notably, several genes had >1.4-fold greater transcript abundance when samples were stored on ice for eight or 24 hours. Our results indicate that RNA degradation and cellular activity of PWBCs have critical impact on transcript abundance when blood samples are stored for 24 hours under refrigeration. As the development of RNA-based biomarkers gain importance in cattle production systems, timely handling of samples is critical for accurate results.

High-throughput RNA sequencing of paraformaldehyde-fixed single cells

Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited. This is because current high-throughput single-cell RNA sequencing methods are either incompatible with or necessitate laborious sample preprocessing for paraformaldehyde treatment, a common tissue and cell fixation and preservation technique. Here we present FD-seq (Fixed Droplet RNA sequencing), a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, permeabilized and sorted single cells. We show that FD-seq preserves the RNA integrity and relative gene expression levels after fixation and permeabilization. Furthermore, FD-seq can detect a higher number of genes and transcripts than methanol fixation. We first apply FD-seq to analyze a rare subpopulation of cells supporting lytic reactivation of the human tumor virus KSHV, and identify TMEM119 as a potential host factor that mediates viral reactivation. Second, we find that infection with the human betacoronavirus OC43 leads to upregulation of pro-inflammatory pathways in cells that are exposed to the virus but fail to express high levels of viral genes. FD-seq thus enables integrating phenotypic with transcriptomic information in rare cell subpopulations, and preserving and inactivating pathogenic samples.

Declining RNA integrity in control autopsy brain tissue is robustly and asymmetrically associated with selective neuronal mRNA signal loss

RNA integrity numbers (RINs) are a standardized method for semi-quantification of RNA degradation, and are used in quality control prior to transcriptional profiling analysis. Recent work has demonstrated that RINs are associated with downstream transcriptional profiling, and correction procedures are typically employed in bioinformatic analysis pipelines to attempt to control for RIN’s influence on gene expression. However, relatively little work has been done to determine whether RIN’s influence is random, or is specifically targeted to a subset of mRNAs. We tested the hypothesis that RIN would be associated with a robust transcriptional profile seen across multiple studies.To test this, we downloaded subsets of raw transcriptional data from six published studies. We only included control, non-pathological post-mortem human brain tissue (n = 383 samples) in which independent subjects’ RIN values were also reported. A robust set of mRNAs consistently and significantly correlated with RIN across multiple studies, appearing to be selectively degraded as RIN declines. Many of the affected gene expression pathways are related to neurons (e.g., vesicle, mRNA transport, synapse, and mitochondria), suggesting that neuronal synaptic mRNA may be particularly vulnerable to degradation. Subsequent analysis of the relationship between RIN and vulnerable mRNA expression revealed most of the decay occurred over a relatively narrow RIN range of 7.2-8.6, with RIN values > 8.6 showing a ceiling effect, and those < 7.2 showing a floor effect on gene expression. Our data suggests that the RIN effect is pathway selective and non-linear, which may be an important consideration for current bioinformatic RIN correcting procedures, particularly in datasets in which declining RIN is confounded with a pathology under study (e.g., in Alzheimer’s disease).

Laser Capture Microdissection optimization for high-quality RNA in mouse brain tissue

Laser Capture Microdissection (LCM) is a method that allows to select and dissecting specific structures, cell populations, or even single cells from different types of tissue to extract DNA, RNA, or proteins. It is easy to perform and precise, avoiding unwanted signals from irrelevant cells, because gene expression may be affected by a bulk of heterogeneous material in a sample. However, despite its efficiency, several steps can affect the sample RNA integrity. In comparison to DNA, RNA is a much more unstable molecule and represents a challenge in the LCM method. Here we describe an optimized protocol to provide good concentration and high-quality RNA in specific structures, such as Dentate Gyrus and CA1 in the hippocampus, basolateral amygdala and anterior cingulate cortex of mouse brain tissue.

How long does the mRNA remains stable in untreated whole bovine blood?

Background: High quality and quantity of messenger RNA (mRNA) are required for accuracy of gene expression studies and other RNA-based downstream applications. Since RNA is considered a labile macromolecular prone to degradation, which may result in falsely altered gene expression patterns, several commercial stabilizing reagents have been developed aiming to keep RNA stable for long period. However, for studies involving large number of experimental samples, the high costs related to these specific reagents may constitute a barrier. Methods and Results: In this context the present study was designed aiming to evaluate the stability of mRNA in whole bovine blood collected in EDTA tubes during storage at common fridge (4°C). Whole blood samples were collected from six Holstein calves and submitted to RNA extraction in each different interval: immediately after blood sampling (< 2 h), at 1-day post-sampling (dps), 2 dps, 3 dps, 7 dps and 14dps intervals. RNA integrity and purity were evaluated, and RT-qPCR assays were run using seven different genes (B2M, ACTB, PPIA, GAPDH, YWHAZ, CD4 and IFN-γ) aiming to evaluate the presence of altered gene transcription during storage. All extracted RNA samples presented high purity, while optimal integrity and unaltered gene expression were observed in whole experimental group up to 3 days of storage.Conclusion: Bovine blood RNA remained stable in K3EDTA tubes for 3 days stored at common fridge and can be successfully and accurately used for gene expression studies.