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.

Integrating Whole Blood Transcriptomic Collection Procedures Into the Current Anti-Doping Testing System, Including Long-Term Storage and Re-Testing of Anti-Doping Samples

Introduction: Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. However, current anti-doping testing does not involve collecting whole blood into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood left over from standard hematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation.Methods: Whole blood samples were collected from twelve and fourteen healthy nonathletic males, for long-term and short-term storage experiments. Long-term storage involved whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., ‒80°C) storage and RNA extracted. Short-term storage involved whole blood collected into K2EDTA tubes and stored at 4°C for 6‒48 h and then incubated at room temperature for 1 and 2 h prior to addition of RNA preservative. RNA quantity, purity, and integrity were analyzed in addition to RNA-Seq using the MGI DNBSEQ-G400 on RNA from both the short- and long-term storage studies. Genes presenting a fold change (FC) of >1.1 or < ‒1.1 with p ≤ 0.05 for each comparison were considered differentially expressed. Microarray analysis using the Affymetrix GeneChip® Human Transcriptome 2.0 Array was additionally conducted on RNA from the short-term study with a false discovery ratio (FDR) of ≤0.05 and an FC of >1.1 or < ‒1.1 applied to identify differentially expressed genes.Results: RNA quantity, purity, and integrity from whole blood subjected to short- and long-term storage were sufficient for gene expression analysis. Long-term storage: when comparing blood tubes with and without RNA preservation 4,058 transcripts (6% of coding and non-coding transcripts) were differentially expressed using microarray and 658 genes (3.4% of mapped genes) were differentially expressed using RNA-Seq. Short-term storage: mean RNA integrity and yield were not significantly different at any of the time points. RNA-Seq analysis revealed a very small number of differentially expressed genes (70 or 1.37% of mapped genes) when comparing samples stored between 6 and 48 h without RNA preservative. None of the genes previously identified in rHuEPO administration studies were differently expressed in either long- or short-term storage experiments.Conclusion: RNA quantity, purity, and integrity were not significantly compromised from short- or long-term storage in blood storage tubes lacking RNA stabilization, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

The Plasmodium NOT1-G paralogue is an essential regulator of sexual stage maturation and parasite transmission

Productive transmission of malaria parasites hinges upon the execution of key transcriptional and posttranscriptional regulatory events. While much is now known about how specific transcription factors activate or repress sexual commitment programs, far less is known about the production of a preferred mRNA homeostasis following commitment and through the host-to-vector transmission event. Here, we show that in Plasmodium parasites, the NOT1 scaffold protein of the CAF1/CCR4/Not complex is duplicated, and one paralogue is dedicated for essential transmission functions. Moreover, this NOT1-G paralogue is central to the sex-specific functions previously associated with its interacting partners, as deletion of not1-g in Plasmodium yoelii leads to a comparable or complete arrest phenotype for both male and female parasites. We show that, consistent with its role in other eukaryotes, PyNOT1-G localizes to cytosolic puncta throughout much of the Plasmodium life cycle. PyNOT1-G is essential to both the complete maturation of male gametes and to the continued development of the fertilized zygote originating from female parasites. Comparative transcriptomics of wild-type and pynot1-g− parasites shows that loss of PyNOT1-G leads to transcript dysregulation preceding and during gametocytogenesis and shows that PyNOT1-G acts to preserve mRNAs that are critical to sexual and early mosquito stage development. Finally, we demonstrate that the tristetraprolin (TTP)-binding domain, which acts as the typical organization platform for RNA decay (TTP) and RNA preservation (ELAV/HuR) factors is dispensable for PyNOT1-G’s essential blood stage functions but impacts host-to-vector transmission. Together, we conclude that a NOT1-G paralogue in Plasmodium fulfills the complex transmission requirements of both male and female parasites.

The Plasmodium NOT1-G Paralogue Acts as an Essential Nexus for Sexual Stage Maturation and Parasite Transmission

Productive transmission of malaria parasites hinges upon the execution of key transcriptional and post-transcriptional regulatory events. While much is now known about how specific transcription factors activate or repress sexual commitment programs, far less is known about the production of a preferred mRNA homeostasis following commitment and through the host-to-vector transmission event. Here we show that Plasmodium parasites have taken the unique approach to duplicate the NOT1 scaffold protein of the CAF1/CCR4/Not complex in order to dedicate one paralogue for essential transmission functions. Moreover, this NOT1-G paralogue is central to the sex-specific functions previously associated with its interacting partners, as deletion of not1-g in Plasmodium yoelii leads to a comparable or complete arrest phenotype for both male and female parasites. We show that, consistent with its role in other eukaryotes, PyNOT1-G localizes to cytosolic puncta throughout much of the Plasmodium life cycle. PyNOT1-G is essential to both the complete maturation of male gametes and to the continued development of the fertilized zygote originating from female parasites. Comparative transcriptomics of wild-type and pynot1-g- parasites shows that loss of PyNOT1-G leads to transcript dysregulation preceding and during gametocytogenesis, and shows that PyNOT1-G acts to preserve mRNAs that are critical to sexual and early mosquito stage development. Finally, we demonstrate that the tristetraprolin-binding domain, which acts as the typical organization platform for RNA decay (TTP) and RNA preservation (ELAV/HuR) factors is dispensable for PyNOT1-Gs essential blood stage functions but impacts host-to-vector transmission. Together, we conclude that Plasmodium has created and adapted a NOT1-G paralogue to fulfill the complex transmission requirements of both male and female parasites.

A Novel HPLC-Based Method to Investigate on RNA after Fixation

RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.

Ethanol as a potential mosquito sample storage medium for RNA preservation

Sample storage for downstream RNA analysis can be challenging in some field settings, especially where access to cryogenic materials or refrigeration/freezer facilities are limited. This has limited RNA-based studies on African malaria vectors collected in the field. We evaluated RNA quality after storing mosquito samples in three different sample preservation media over a 4-week period. Storing mosquito specimens in cold (4°C) media significantly improved yields of intact RNA. Our results indicate commercially available products perform well in keeping RNA integrity as advertised. Moreover, absolute ethanol may be an economical alternative for sample preservation that can be utilized in some resource-limited settings.