Project description:Study to detect possible interactions between blood collection tubes and RNA purification methods on extracellular RNA (exRNA) sequencing. Different combinations of blood collection tubes (3 in total), time intervals between blood draw and downstream processing (immediate (T0), after 4 hours (T4), after 16 hours (T16)), and RNA purification kits (2 in total) were evaluated by applying mRNA capture sequencing (Illumina) to exRNA from human healthy donor plasma or serum.
Project description:Study to detect possible interactions between blood collection tubes and RNA purification methods on extracellular RNA (exRNA) sequencing. Different combinations of blood collection tubes (3 in total), time intervals between blood draw and downstream processing (immediate (T0), after 4 hours (T4), after 16 hours (T16)), and RNA purification kits (2 in total) were evaluated by applying Small RNA sequencing (Illumina) to exRNA from human healthy donor plasma or serum.
Project description:To evaluate the impact of blood collection tubes on extracellular RNA (exRNA) sequencing, 10 different blood collection tubes were compared by applying RNA Exome sequencing (Illumina) to exRNA from human healthy donor plasma or serum. Three time spans between blood draw and downstream processing were evaluated for each of the tubes. Preservation tubes were processed immediately upon blood collection (T0), after 24 hours (T24), or after 72 hours (T72). Non-preservation plasma and serum tubes were processed immediately upon blood collection (T0), after 4 hours (T4), or after 16 hours (T16). Due to donor privacy concerns the raw data for this study have been submitted to the controlled-access archive EGA under the accession EGAS00001005263.
Project description:To evaluate the impact of blood collection tubes on extracellular RNA (exRNA) sequencing, 10 different blood collection tubes were compared by applying Small RNA sequencing (Illumina) to exRNA from human healthy donor plasma or serum. Three time spans between blood draw and downstream processing were evaluated for each of the tubes. Preservation tubes were processed immediately upon blood collection (T0), after 24 hours (T24), or after 72 hours (T72). Non-preservation plasma and serum tubes were processed immediately upon blood collection (T0), after 4 hours (T4), or after 16 hours (T16). Due to donor privacy concerns the raw data for this study have been submitted to the controlled-access archive EGA under the accession EGAS00001005263.
Project description:To evaluate the impact of the RNA purification method on extracellular RNA (exRNA) sequencing, 8 different RNA purification kits were compared by applying RNA Exome sequencing (Illumina) to exRNA from human healthy donor plasma. Minimum and maximum plasma input volumes recommended by the manufacturers were tested in triplicate.
Project description:Extracellular RNAs (exRNAs) in blood and other biofluids have attracted great interest as potential biomarkers in liquid biopsy applications, as well as for their potential biological functions. Whereas it is well-established that extracellular microRNAs are present in human blood circulation, the degree to which messenger RNAs (mRNA) and long noncoding RNAs (lncRNA) are represented in plasma is less clear. Here we report that mRNA and lncRNA species are present as small fragments in plasma that are not detected by standard small RNA-seq methods, because they lack 5’-phosphorylation or carry 3’-phosphorylation. We developed a modified sequencing protocol (termed “phospho-sRNA-seq”) that incorporates upfront RNA treatment with T4 polynucleotide kinase (which also has 3’ phosphatase activity) and compared it to a standard small RNA-seq protocol, using as input both a pool of synthetic RNAs with diverse 5’ and 3’ end chemistries, as well exRNA isolated from human blood plasma. This series uses the synthetic pools of small RNAs to demonstrate the efficacy of phospho-sRNA-seq to enable capture of sRNAs lacking a 5’ phosphate and/or having a 3’ phosphate.
Project description:To evaluate the impact of the RNA purification method on extracellular RNA (exRNA) sequencing, 8 different RNA purification kits were compared by applying Small RNA sequencing (Illumina) to exRNA from human healthy donor plasma. Minimum and maximum plasma input volumes recommended by the manufacturers were tested in triplicate. Due to donor privacy concerns the raw data for this study have been submitted to the controlled-access archive EGA under the accession EGAS00001005263.
Project description:The extracellular RNAs (exRNAs) from human biofluid have recently been systematically characterized. However, the correlations of biofluid exRNA levels and human diseases remain largely untested. Here, considering the unmet need for presymptomatic biomarkers of sporadic Alzheimer’s disease (AD), we leveraged the recently developed SILVER-seq (small-input liquid volume extracellular RNA sequencing) technology to generate exRNA profiles from a longitudinal collection of human plasma samples. These 164 plasma samples were collected from research subjects 70 years or older with up to 15 years of clinical follow-up prior to death and whose clinical diagnoses were confirmed by pathological analysis of their post mortem brains. The exRNAs of AD-activated genes and transposons in the brain exhibited a concordant trend of increase in AD plasma in comparison with age-matched control plasma. However, when we required statistical significance with multiple testing adjustments, phosphoglycerate dehydrogenase (PHGDH) was the only gene that exhibited consistent upregulation in AD brain transcriptomes from 3 independent cohorts and an increase in AD plasma as compared to controls. We validated PHGDH’s serum exRNA and brain protein expression increases in AD by using 5 additional published cohorts. Finally, we compared the time-course exRNA trajectories between “converters” and controls. Plasma PHGDH exRNA exhibited presymptomatic increases in each of the 11 converters during their transitions from normal to cognitive impairment but remained stable over the entire follow-up period in 8 out of the 9 control elderly subjects. These data suggest the potential utilities of plasma exRNA levels for screening and longitudinal exRNA changes as a presymptomatic indication of sporadic AD.
