Journal: bioRxiv

Article Title: Performance of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study

doi: 10.1101/2021.05.11.442610

Figure Lengend Snippet: Overview of the exRNAQC phase 1 study design. To evaluate the impact of the 8 RNA purification methods (left panel), two blood draws from a single individual were performed to separately apply mRNA capture (study code: exRNAQC004) and small RNA (study code: exRNAQC011) sequencing. Both minimum and maximum recommended plasma input volumes were tested in triplicate. To compare RNA purification performance, 9 performance metrics were calculated. To evaluate the impact of the 10 blood collection tubes (right panel), 9 individuals were sampled, enabling to test 3 time intervals between blood draw and downstream processing 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). Both mRNA capture (study code: exRNAQC005) and small RNA (study code: exRNAQC013) sequencing were performed, and the data was analyzed using 5 performance metrics. To control the RNA purification and library preparation workflows, 189 synthetic spike-in RNA molecules (Sequin and Extracellular RNA Communication Consortium (ERCC) spike-ins for RNA Exome sequencing, and RNA extraction Control (RC) and Library Prep Control (LP) spike-ins for Small RNA sequencing) were used, allowing to calculate relative RNA concentrations and purification efficiency (lower panel). ACD-A: BD Vacutainer Glass ACD Solution A tube; ALC: area left of the curve; Biomatrica: LBgard Blood Tube; CCF: QIAamp ccfDNA/RNA Kit; CIRC: Plasma/Serum Circulating and Exosomal RNA Purification Kit/Slurry Format; citrate: Vacuette Tube 9 ml 9NC Coagulation sodium citrate 3.2%; DNA Streck: Cell-Free DNA BCT; EDTA: BD Vacutainer Plastic K2EDTA tube; EDTA separator: Vacuette Tube 8 ml K2E K2EDTA Separator; MAP: MagNA Pure 24 Total NA Isolation Kit in combination with the MagNA Pure instrument; MAX: the Maxwell RSC miRNA Plasma and Exosome Kit in combination with the Maxwell RSC Instrument; MIR: the miRNeasy Serum/Plasma Kit; MIRA: the miRNeasy Serum/Plasma Advanced Kit; MIRV: the mirVana PARIS Kit with purification protocol for total RNA; MIRVE: mirVana PARIS Kit with purification protocol for RNA enriched for small RNAs; NUC: the NucleoSpin miRNA Plasma Kit; PAXgene: PAXgene Blood ccfDNA Tube; RNA Streck: Cell-Free RNA BCT; Roche: Cell-Free DNA Collection Tube; serum: BD Vacutainer SST II Advance Tube.

Article Snippet: In total, 10 different blood collection tubes were used: the BD Vacutainer SST II Advance Tube (referred to as serum in this study; Becton Dickinson and Company, 366444), BD Vacutainer Plastic K2EDTA tube (EDTA; Becton Dickinson and Company, 367525), Vacuette Tube 8 ml K2E K2EDTA Separator (EDTA separator; Greiner Bio-One, 455040), BD Vacutainer Glass ACD Solution A tube (ACD-A; Becton Dickinson and Company, 366645), Vacuette Tube 9 ml 9NC Coagulation sodium citrate 3.2% (citrate; Greiner Bio-One, 455322), Cell-Free RNA BCT (RNA Streck; Streck, 230248), Cell-Free DNA BCT (DNA Streck; Streck, 218996), PAXgene Blood ccfDNA Tube (PAXgene; Qiagen, 768115), Cell-Free DNA Collection Tube (Roche; Roche, 07785666001), and LBgard Blood Tube (Biomatrica; Biomatrica, M68021-001).

Techniques: Purification, Sequencing, Clinical Proteomics, Preserving, Control, RNA Extraction, RNA Sequencing, Coagulation, Isolation

Journal: bioRxiv

Article Title: Performance of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study

doi: 10.1101/2021.05.11.442610

Figure Lengend Snippet: Summary of mean fold changes (FC) between time point 1 (centrifugation step 4 hours after blood collection for non-preservation tubes; 24 hours for preservation tubes) and time point 0 vs. time point 2 (centrifugation step 16 hours after blood collection for non-preservation tubes; 72 hours for preservation tubes) and time point 0, per tube and per metric, for mRNA capture sequencing ( left ) and small RNA sequencing ( right ). Ideally, the mean FC of the stability metrics approaches 1, indicating that there is little change from baseline and the blood collection tube performs well across time. Legend: “gene/miRNA count” represents stability of the absolute number of protein coding genes (mRNA) or absolute number of miRNAs (small RNA), “RNA concentration” corresponds to the stability of the relative RNA concentration as determined by number of endogenous reads vs Sequin spike-in RNA (mRNA) or the stability of the relative RNA concentration as determined by number of endogenous reads vs RC spike-in RNA (small RNA), “hemolysis” corresponds to stability of the absorbance of light at 414 nm (mRNA and small RNA), “biotype” corresponds to the stability of the fraction of all counts mapping to mRNAs (i.e. the protein coding fraction) or the stability of the fraction of all counts mapping to micro RNAs (small RNA), “ALC” corresponds to the area left of the curve, a reproducibility metric (mRNA and small RNA). Non-preservation tubes are the BD Vacutainer Plastic K2EDTA tube (EDTA), Vacuette Tube 8 ml K2E K2EDTA Separator (EDTA separator), BD Vacutainer Glass ACD Solution A tube (ACD-A), Vacuette Tube 9 ml 9NC Coagulation sodium citrate 3.2% (citrate), and BD Vacutainer SST II Advance Tube (serum). The preservation tubes are the Cell-Free RNA BCT (RNA Streck), Cell-Free DNA BCT (DNA Streck), PAXgene Blood ccfDNA Tube (PAXgene), Cell-Free DNA Collection Tube (Roche) and LBgard Blood Tube (Biomatrica). Note that different donors were sampled and that tubes were processed at different time points for preservation and non-preservation tubes.

Article Snippet: In total, 10 different blood collection tubes were used: the BD Vacutainer SST II Advance Tube (referred to as serum in this study; Becton Dickinson and Company, 366444), BD Vacutainer Plastic K2EDTA tube (EDTA; Becton Dickinson and Company, 367525), Vacuette Tube 8 ml K2E K2EDTA Separator (EDTA separator; Greiner Bio-One, 455040), BD Vacutainer Glass ACD Solution A tube (ACD-A; Becton Dickinson and Company, 366645), Vacuette Tube 9 ml 9NC Coagulation sodium citrate 3.2% (citrate; Greiner Bio-One, 455322), Cell-Free RNA BCT (RNA Streck; Streck, 230248), Cell-Free DNA BCT (DNA Streck; Streck, 218996), PAXgene Blood ccfDNA Tube (PAXgene; Qiagen, 768115), Cell-Free DNA Collection Tube (Roche; Roche, 07785666001), and LBgard Blood Tube (Biomatrica; Biomatrica, M68021-001).

Techniques: Centrifugation, Preserving, Sequencing, RNA Sequencing, Concentration Assay, Coagulation

Journal: bioRxiv

Article Title: Performance of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study

doi: 10.1101/2021.05.11.442610

Figure Lengend Snippet: a: boxplot of the fold change within each donor across time points, per tube, for hemolysis in platelet-free plasma, as measures by absorbance at 414 nm with Nanodrop; b: boxplot of fold change of relative RNA concentration, based on the ratio of endogenous reads vs Sequin spike-in RNA reads; c: boxplot of the fold change of the number of genes, after filtering genes with counts fewer than 6 reads; d: area left of the curve, transformed from log2 to linear scale; e: boxplot of the fold change of the fraction of the counts mapping to protein coding genes vs. all counts. The white triangle on the boxplot corresponds to the mean of the fold change. 1 st time point corresponds to the comparison of T04 vs. T0 (non-preservation tubes: BD Vacutainer Plastic K2EDTA tube (EDTA), Vacuette Tube 8 ml K2E K2EDTA Separator (EDTA separator), BD Vacutainer Glass ACD Solution A tube (ACD-A), Vacuette Tube 9 ml 9NC Coagulation sodium citrate 3.2% (citrate), and BD Vacutainer SST II Advance Tube (serum)) or T24 vs. T0 (preservation tubes: Cell-Free RNA BCT (RNA Streck), Cell-Free DNA BCT (DNA Streck), PAXgene Blood ccfDNA Tube (PAXgene), Cell-Free DNA Collection Tube (Roche) and LBgard Blood Tube (Biomatrica)). 2 nd time point corresponds to the comparison of T16 vs. T0 (non-preservation tubes) or T72 vs T0 (preservation tubes). T0: plasma prepared immediately after blood draw. T04, T16, T24, T72: plasma prepared 4 hours, 16 hours, 24 hours and 72 hours after blood draw, respectively. Note that different donors were sampled and that tubes were processed at different time points for preservation and non-preservation tubes.

Article Snippet: In total, 10 different blood collection tubes were used: the BD Vacutainer SST II Advance Tube (referred to as serum in this study; Becton Dickinson and Company, 366444), BD Vacutainer Plastic K2EDTA tube (EDTA; Becton Dickinson and Company, 367525), Vacuette Tube 8 ml K2E K2EDTA Separator (EDTA separator; Greiner Bio-One, 455040), BD Vacutainer Glass ACD Solution A tube (ACD-A; Becton Dickinson and Company, 366645), Vacuette Tube 9 ml 9NC Coagulation sodium citrate 3.2% (citrate; Greiner Bio-One, 455322), Cell-Free RNA BCT (RNA Streck; Streck, 230248), Cell-Free DNA BCT (DNA Streck; Streck, 218996), PAXgene Blood ccfDNA Tube (PAXgene; Qiagen, 768115), Cell-Free DNA Collection Tube (Roche; Roche, 07785666001), and LBgard Blood Tube (Biomatrica; Biomatrica, M68021-001).

Techniques: Clinical Proteomics, Concentration Assay, Transformation Assay, Comparison, Preserving, Coagulation

Journal: bioRxiv

Article Title: Performance of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study

doi: 10.1101/2021.05.11.442610

Figure Lengend Snippet: a: boxplot of the fold change within each donor across time points, per tube, for hemolysis in platelet-free plasma, as measures by absorbance at 414 nm with Nanodrop; b: boxplot of fold change of relative RNA concentration, based on the ratio of endogenous reads vs RC spike-in RNA reads; c: boxplot of the fold change of the number of micro RNAs, after filtering miRNAs with counts fewer than 3 reads; d: area left of the curve, transformed from log2 to linear scale; e: boxplot of the fold change of the fraction of the counts mapping to micro RNAs vs. all counts. The white triangle on the boxplot corresponds to the mean of the fold change. 1 st time point corresponds to the comparison of T04 vs. T0 (non-preservation tubes: BD Vacutainer Plastic K2EDTA tube (EDTA), Vacuette Tube 8 ml K2E K2EDTA Separator (EDTA separator), BD Vacutainer Glass ACD Solution A tube (ACD-A), Vacuette Tube 9 ml 9NC Coagulation sodium citrate 3.2% (citrate), and BD Vacutainer SST II Advance Tube (serum)) or T24 vs. T0 (preservation tubes: Cell-Free RNA BCT (RNA Streck), Cell-Free DNA BCT (DNA Streck), PAXgene Blood ccfDNA Tube (PAXgene), Cell-Free DNA Collection Tube (Roche) and LBgard Blood Tube (Biomatrica)). 2 nd time point corresponds to the comparison of T16 vs. T0 (non-preservation tubes) or T72 vs T0 (preservation tubes). T0: plasma prepared immediately after blood draw. T04, T16, T24, T72: plasma prepared 4 hours, 16 hours, 24 hours and 72 hours after blood draw, respectively. Note that different donors were sampled and that tubes were processed at different time points for preservation and non-preservation tubes.

Article Snippet: In total, 10 different blood collection tubes were used: the BD Vacutainer SST II Advance Tube (referred to as serum in this study; Becton Dickinson and Company, 366444), BD Vacutainer Plastic K2EDTA tube (EDTA; Becton Dickinson and Company, 367525), Vacuette Tube 8 ml K2E K2EDTA Separator (EDTA separator; Greiner Bio-One, 455040), BD Vacutainer Glass ACD Solution A tube (ACD-A; Becton Dickinson and Company, 366645), Vacuette Tube 9 ml 9NC Coagulation sodium citrate 3.2% (citrate; Greiner Bio-One, 455322), Cell-Free RNA BCT (RNA Streck; Streck, 230248), Cell-Free DNA BCT (DNA Streck; Streck, 218996), PAXgene Blood ccfDNA Tube (PAXgene; Qiagen, 768115), Cell-Free DNA Collection Tube (Roche; Roche, 07785666001), and LBgard Blood Tube (Biomatrica; Biomatrica, M68021-001).

Techniques: Clinical Proteomics, Concentration Assay, Transformation Assay, Comparison, Preserving, Coagulation