Biology subjects

De Hoff, P.

Publications and source records attributed to De Hoff, P..

2 recordsLinked to original sources

bioRxiv2025

Miniaturized Workflow for Transcriptomic Profiling of Urinary Extracellular RNA during Pregnancy

Urine contains extracellular RNA (exRNA) carried by extracellular vesicles (EVs) and other biomolecular complexes. There is currently a need for studies focused on female cohorts to develop new methods for non-invasive analysis of biofluids to create reference profiles and for identification of biomarkers of reproductive and pregnancy disorders. The objective of this study was therefore to identify optimal methods for transcriptomic profiling of urine by testing different exRNA isolation and scalable library preparation methods that enable detection of biomarkers that reflect pregnancy-associated changes in the placenta and maternal tissues. RNA was extracted from pooled and individual urine samples obtained from normal non-pregnant and pregnant females, as well as males, using input volumes of either 0.6 mL, 1 mL, or 4 mL. Samples were extracted using methods that focused either on isolating vesicular (EV-associated) or total (EV-associated and non EV-associated) exRNA. Small RNA libraries (n=208) were prepared using the NEBNext Small RNA Library Prep kit and long RNA libraries (n=97) were prepared using the SMART-Seq v4 Ultra Low Input RNA or the SMARTer Stranded Total RNA-Seq Kit v2 Pico Input kits (Takara). Principal component analysis showed that the greatest source of variance amongst technical replicates of small RNA libraries (n=176 which passed quality control) was exRNA isolation method, and amongst long RNA libraries (n=97 which passed quality control) was library preparation method. Long RNA libraries prepared from exRNA extracted using miRCURY showed that the SMART-Seq v4 method yielded significantly more uniquely mapped reads compared to the Pico v2 method (p<0.05). We have established a scalable pipeline for small and long RNA-Seq profiling of exRNA in urine in a reproducible manner, which we used to identify differentially expressed urinary exRNAs in pregnancy, and will enable transcriptomic profiling of urinary exRNA in disorders of pregnancy, including preeclampsia.

genetics↗

bioRxiv2022

Sentinel Cards Provide Practical SARS-CoV-2 Monitoring in School Settings

Accurate, high-resolution environmental monitoring of SARS-CoV-2 traces indoors through sentinel cards is a promising approach to help students safely return to in-person learning. Because SARS-CoV-2 RNA can persist for up to a week on several indoor surface types, there is a need for increased temporal resolution to determine whether consecutive surface positives arise from new infection events or continue to report past events. Cleaning sentinel cards after sampling would provide the needed resolution, but might interfere with assay performance. We tested the effect of three cleaning solutions (BZK wipes, wet wipes, RNase Away) at three different viral loads: "high" (4 x 104 GE/mL), "medium" (1 x 104 GE/mL), and "low" (2.5 x 103 GE/mL). RNAse Away, chosen as a positive control, was the most effective cleaning solution on all three viral loads. Wet wipes were found to be more effective than BZK wipes in the medium viral load condition. The low viral load condition was easily reset with all three cleaning solutions. These findings will enable temporal SARS-CoV-2 monitoring in indoor environments where transmission risk of the virus is high and the need to avoid individual-level sampling for privacy or compliance reasons exists. ImportanceBecause SARS-CoV-2, the virus that causes COVID-19, persists on surfaces, testing swabs taken from surfaces is useful as a monitoring tool. This approach is especially valuable in school settings, where there are cost and privacy concerns that are eliminated by taking a single sample from a classroom. However, the virus persists for days to weeks on surface samples, so it is impossible to tell whether positive detection events on consecutive days are persistent signal or new infectious cases, and therefore whether the positive individuals have been successfully removed from the classroom. We compare several methods for cleaning "sentinel cards" to show that this approach can be used to identify new SARS-CoV-2 signals day to day. The results are important for determining how to monitor classrooms and other indoor environments for SARS-CoV-2 virus.

bioinformatics↗