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.

Isolation of High Quality RNA for High Throughput Applications From Secondary Metabolite Rich Crocus Sativus L

Isolating high quality RNA is a basic requirement while performing high throughput sequencing, microarray and various other molecular investigations. However, it has been quite challenging to isolate RNA with absolute purity from plants like Crocus sativus that are rich in secondary metabolites, polysaccharides and other interfering compounds which often irreversibly co-precipitate with the RNA. While many methods have been proposed for RNA extraction that include CTAB, TriZol, SDS based methods, they invariably yield less and poor quality RNA. In the present study we made certain changes in the available protocols including modifications in the extraction buffer and procedure viz-a-viz solutions used for precipitation of RNA. Our method led to the isolation of clear and non-dispersive total RNA with an RNA Integrity Number (RIN) greater than 7.5. The quality of the RNA was further assessed by qPCR based amplification of mature miRNAs such as Cs-MIR166c and Cs- MIR396a. In conclusion, the study describes an efficient method of RNA extraction that is highly ideal for high throughput sequencing of small RNAs.

Protocols for the Extraction of High-quality RNA from Pineapple Tiller, Flower, Inflorescence, and Fruits

High-quality RNA is an important genetic study as it has minimal contaminants that can affect gene discovery including degraded RNAs, chemical, and biological residues. Hence, it is a prerequisite for genetic analysis using Next Generation Sequencing (NGS) for accurate and reliable data mining. Despite its importance, extracting high-quality RNA from different samples is often a challenge, as every tissue has a different biochemical composition, thus requiring different protocols. This paper reports protocols for the extraction of high-quality RNA from two type of pineapple tissues, which are thickly lignified hard tissue (tillers, inflorescence, flowers) and watery soft tissue (mature fruit, ripe fruit, and overripe fruit) via modified Kim and Hamada (2005) method. Total RNA was extracted in all six tissues, which showed two distinctive 25S and 18S band on agarose gel. The total RNA in this study was considered high-quality as the minimum concentration was 50 ng/μl, the absorbance ratio (A260:A280) was more than 1.8 and RNA integrity number (RIN) was greater than 7. The obtained results showed that the modified Kim and Hamada (2005) method was effective in extracting high-quality RNA from the challenging MD2 pineapple tissue, which is suitable for subsequent molecular analysis, including the highly sensitive NGS.

Do Tissues Fixed in a Non-cross-linking Fixative Require a Dedicated Formalin-free Processor?

We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF−ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF−ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9–2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF−ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem:

A diagnostic relationship between the RNA Integrity Number equivalent and Time Since Deposition of blood

Determining the age, or time since deposition (TSD), of bloodstains would provide forensic scientists with critical information regarding the timeline of the events of bloodshed during a crime. The physicochemical changes that occur to major biomolecules as a bloodstain dries can be used to estimate the TSD of bloodstains. For example, high-resolution automated gel electrophoresis can be used to quantify the timewise degradation of DNA present in bloodstains. Our study aims to analyze and quantify the timewise degradation trends found in total RNA from bloodstains, expanding the scope of the TSD research which has previously explored DNA and targeted mRNA molecules. Fifty bloodstains were stored in plastic microcentrifuge tubes at room temperature and tested over 10 different timepoints spanning one week. A total of eight RNA metrics were visually assessed and quantified using linear regression. RNA Integrity Number equivalent (RINe), total RNA concentration, and 28S/18S rRNA peak area ratios were retained for further analyses based on their relationship with time and limited correlations. RINe and total RNA concentration both exhibited negative trends over time, highlighting a decrease in quality and quantity. RINe was the RNA metric that demonstrated the greatest association with time (R2 = 0.696). Generalized linear mixed-effects models including donor (biological replicate) as a random effect increased the fit for all RNA metrics to varying degrees, but no significant differences were found between biological replicates for the RINe metric. Our results illustrated the presence of a significant decrease in the retained RNA metrics after 24 hours, suggesting that this method could be used to reliably differentiate day-old bloodstains from older bloodstains. Future work should focus on recreating this study in different environmental conditions, including testing on a variety of substrates.

homeRNA: A self-sampling kit for the collection of peripheral blood and stabilization of RNA

ABSTRACTGene expression analysis (e.g., targeted small gene panels, transcriptomics) from whole blood can elucidate mechanisms of immune function and aid in the discovery of biomarkers. Conventional in-clinic venipuncture offers only a small snapshot of our broad immune landscape as immune responses may occur outside of the time and location parameters available for conventional venipuncture. A self-operated method that enables flexible sampling of liquid whole blood coupled with an immediate stabilization of cellular RNA is instrumental in facilitating capture and preservation of acute or transient immune fluxes. To this end, we developed homeRNA: a kit that allows for self-collection of peripheral blood (∼0.5 mL) and immediate stabilization of cellular RNA, using the Tasso-SST™ blood collection device paired with a specially designed stabilizer tube containing RNAlater™. To assess the usability and feasibility of homeRNA for self-collection and stabilization of whole blood RNA, we conducted a pilot study (n = 41 participants) where we sent homeRNA to participants aged 21-69, located across 10 US states (94% successful blood collections, n = 51). Among participants who successfully collected blood, 91% reported no or minimal pain/discomfort using the kit (n = 35), and 77% reported easy or somewhat easy stabilization protocol. Total RNA yield from the stabilized samples ranged between 0.24 µg and 5.99 µg (mean = 1.65 µg), while RNA Integrity Number (RIN) values were above 7.0 (mean = 7.9), indicating limited RNA degradation. Results from this study demonstrate the self-collection and RNA stabilization of whole blood with homeRNA by participants themselves, in their own home.

The spatial RNA integrity number assay for in situ evaluation of transcriptome quality

The RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows.

Extraction of High-quality RNA from Metabolite and Pectin Rich Recalcitrant Calyx Tissue of Hibiscus sabdariffa L.

Hibiscus sabdariffa L. is no stranger to the field of pharmacology, as its calyx extract is highly rich in beneficial compounds and has been demonstrated to possess antihyperglycemic, antihypertension, anticancer and antioxidant properties. Thus, it is labeled it as a functional food with great health benefits and therapeutic potentials. The medicinal and nutritional components of the calyx are well reported. On the contrary, not much is known about the molecular machineries governing the biosynthesis of beneficial compounds in this plant. Obtaining good yields of high-quality RNA is crucial for the success of downstream research pertaining to molecular biology. However, the presence of high quantities of phenolic compounds, polysaccharides, mucilage and pectin in the fibrous calyx tissue poses major challenges for RNA extraction in H. sabdariffa. Here, we modified a CTAB-based method for efficient extraction of high-quality RNA from the calyx tissue. High quality RNA samples having RNA integrity number of more than eight were successfully extracted. The purities of RNA samples were also confirmed by the A260/280 and A260/230 values. Subsequent successful preparation of a sequencing library using one of the RNA samples extracted via the modified CTAB method further emphasized the efficiency of this extraction protocol and quality of the RNA samples. The results showed that the modified CTAB method was effective in extracting good quality RNA from the challenging calyx tissue of Hibiscus sabdariffa L. suitable for sensitive downstream application.

46 Endometrial transcriptome profiles associated with bovine preimplantation pregnancy outcome and parity

Pregnancy failure in dairy cattle appears to be attributable to losses during embryogenesis, mainly within the first month. Models have estimated that pregnancies lost after day 30 of fertilization cost producers US $550/cow. The reasons for these losses are not well understood, and the transcriptome profile of the reproductive tract during the establishment of pregnancy may point to mechanisms. Thirty-eight cows in lactations 1 to 3 were artificially inseminated, and endometrial biopsies were collected on day 15 after artificial insemination to investigate the effect of parity on the preimplantation pregnancy outcome. Pregnancy status was determined by the presence of interferon-tau in the uterine flushing, revealing 19 pregnant (P) and 19 non-pregnant (NP) cows. Nineteen biopsy samples [9 cows in lactation 1 (6 P and 3 NP), 4 in lactation 2 (2 P and 2 NP), and 6 in lactation 3 (3P and 3NP)] with an average RNA integrity number of 7 were selected for RNA sequencing with an Illumina HiSeq analyzer. Sequence reads were assembled to the ARS_USD1.2.99 bovine reference genome using the CLC genomics workbench software. On average, the samples generated ~56 million reads and 94.99% were mapped to the reference genome. Differential gene expression analysis between P and NP cows identified 187, 60, and 136 differentially expressed genes (DEG) in lactation 1, 2 and 3, respectively (P < 0.01, FDR < 0.05, FC > ±2). Metabolic pathway enrichment analysis identified several DEG upregulated in lactations 2 and 3 associated with IL-17 signaling pathway, although expression of IL-17 itself was not different. This pathway is part of the protective response against extracellular bacteria and likely participates in pregnancy maintenance through local regulation of immune function. Further functional genomic analyses will be performed to determine additional metabolic pathways, key regulator genes, and functional SNPs associated with the establishment of pregnancy.