From Sampling to Sequencing: A Liquid Biopsy Pre-Analytic Workflow to Maximize Multi-Layer Genomic Information from a Single Tube.
Author(s): Maass KK, Schad PS, Finster AME, Puranachot P, Rosing F, Wedig T, Schwarz N, Stumpf N, Pfister SM, Pajtler KW
Publication: Cancers (Basel), 2021, Vol. 13, Page
PubMed ID: 34203921 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate the effects of collection tube type, storage duration, and cell-free DNA (cfDNA) isolation method used on the cfDNA and cell-free RNA (cfRNA) yield and purity from cerebrospinal fluid (CSF), blood, and plasma.
Conclusion of Paper
The largest plasma volume and highest yield of 160 bp DNA was obtained when blood was collected in Norgen tubes but more copies of RPP30 were found when collected in EDTA tubes. cfDNA yield was lower when blood was stored in preservative tubes for 7 days than 3 days. cfDNA yields were highest when extraction was with NucleoSnap or the QIAamp MinElute ccfDNA Mini Kit. DNA purity and methylation profiles were comparable in all specimens, regardless of collection tube. Bioanalyzer analysis showed that size selection using magnetic beads successfully eliminated gDNA while retaining the cfDNA, resulting in an increase in the cfDNA fraction from 81% to 95%. cfRNA concentrations were higher when extraction was with NucleoSpin rather than NucleoSnap but were lower from Streck plasma than other tube types and when blood was stored for 5 days rather than 3 days.
DNA yields were low to undetectable in the CSF of the four healthy patients, depending on collection tube type. cfDNA and cfRNA yield increased when PBS was added to Norgen, Streck, and PAXgene tubes prior to centrifugation but this increase was only observed in all tube types when analyzed by ddPCR.
Studies
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Study Purpose
The purpose of this study was to compare the cfDNA yield and purity and cfRNA yield from EDTA plasma and blood from four healthy patients stored in Norgen cf-DNA/cf-RNA Preservative Tubes, PAXgene Blood ccfDNA Tube IVD, and Streck Cell-Free DNA BCT for 3 or 7 days. The cfDNA and cfRNA yield using the NucleoSpin and NucleoSnap kits were also compared. Blood was collected from four healthy patients in duplicate into EDTA tubes, Norgen cf-DNA/cf-RNA Preservative Tubes, PAXgene Blood ccfDNA Tube IVD, and Streck Cell-Free DNA BCT. Plasma was obtained from blood in EDTA tubes by centrifugation of blood at 1900 x g for 10 min at 4°C within 1 h of collection. Plasma was obtained from blood in the other tube types 3 days and 7 days after collection by room temperature centrifugation at 500 x g for 20 min (Norgen), 1900 x g for 15 min (PAXgene), or 1600 x g for 15 min (Streck). cfDNA and cfRNA were isolated from plasma using the NucleoSpin and NucleoSnap kits. cfDNA was quantified by bioanalyzer (160 bp DNA) and total DNA was quantified by bioanalyzer (50-7000 bp DNA) and Qubit. cfDNA levels were also assessed by ddPCR amplification of RPP30. cfRNA was quantified by Qubit. Methylation profiles were assessed in the plasma of one patient using EPIC array.
Summary of Findings:
Plasma volume was significantly higher when blood was collected into Norgen or PAXgene tubes than EDTA tubes (P<0.0001 and P=0.0021, respectively) and significantly lower when collected into Streck tubes compared to EDTA, PAXgene, and Norgen tubes (P<0.0001). Yield of 160 bp DNA was highest when blood was collected in Norgen followed by EDTA, PAX, and Streck tubes but the differences in yields per mL plasma were small with highest yields per mL from EDTA tubes and the lowest from PAXgene tubes. ddPCR amplification of RPP30 found the highest genome equivalents per tube when blood was collected in EDTA followed by PAXgene tubes but significance of differences was dependent on isolation method and storage duration. The purity of the cfDNA (ratio 146 to 176 bp DNA to 50-7000 bp DNA) was close to one in all specimens, indicating a low level of genomic contamination in all specimens, regardless of collection tube type. cfDNA yield was slightly lower from blood stored in preservative tubes for 7 days than the specimen stored in the same type of preservative tube stored for 3 days but total DNA levels were equivalent between the two timepoints for Norgen and PAXgene tubes, indicating increased leukocyte DNA contamination with processing delay in these tubes. More cfDNA was obtained when extraction was with NucleoSnap than NucleoSpin, regardless of collection tube type and storage duration. Methylation profiles were similar among plasma specimens from a single patient stored for 7 days in each of the different tube types and the fresh EDTA specimen. cfRNA yield was low from Streck plasma, regardless of extraction method. cfRNA concentrations were higher when extraction was with NucleoSpin rather than NucleoSnap (P<0.0001) and for all three preservative tube the yields were lower when blood was stored 5 days rather than 3 days.
Biospecimens
Preservative Types
- Other Preservative
- None (Fresh)
- Streck/BCT
- PAXgene
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Bisulfite conversion assay DNA Automated electrophoresis/Bioanalyzer DNA Digital PCR RNA Automated electrophoresis/Bioanalyzer RNA Digital PCR RNA Fluorometry DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Norgen tube
PAXgene tube
Streck tube
EDTA tube
Biospecimen Preservation Type of fixation/preservation Blood collection tube additive
EDTA
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Digital PCR Specific Targeted nucleic acid RPP30
Analyte Extraction and Purification Analyte isolation method NucleoSnap
NucleoSpin
Fluorometry Specific Technology platform Qubit
Bioanalyzer
TapeStation
Storage Storage duration 0 days
3 days
7 days
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Study Purpose
The purpose of this study was to compare the cfDNA yield and purity in CSF from healthy patients and a patient with a brain tumor stored in Norgen cf-DNA/cf-RNA Preservative Tubes, PAXgene Blood ccfDNA Tube IVD, and Streck Cell-Free DNA BCT for 7 days. The effect of filling the tube with PBS prior to centrifugation was also investigated. CSF was collected by lumbar puncture from four healthy patients and one brain cancer patient in duplicate into plain tubes, Norgen cf-DNA/cf-RNA Preservative Tubes, PAXgene Blood ccfDNA Tube IVD, and Streck Cell-Free DNA BCT and stored for 7 days at 4°C. Before centrifugation, one of each tube type was filled to the top with PBS while the other was left without PBS. CSF was centrifuged (speed, time and temperature not included). cfDNA and cfRNA were isolated from plasma using the NucleoSpin kit. cfDNA (160 bp DNA) and total DNA (50-7000 bp DNA) were quantified by bioanalyzer. cfRNA and cfDNA were quantified by ddPCR amplification of RPP30.
Summary of Findings:
DNA yields were low from CSF of the four healthy patients and no cfDNA peaks were observed, regardless of tube type. Addition of the manufacturer recommended PBS to the CSF from a brain tumor patient in Norgen tubes increased the yield of DNA significantly but this was not found for the other tube types. cfDNA was only detected by bioanalyer in CSF in Norgen tubes after addition of PBS and in plain tubes, but the specimen in Norgen tubes with PBS had more detectable cfDNA and less contamination from genomic DNA. ddPCR analysis showed an increase in both cfDNA and cfRNA yield when PBS was added to each of the preservative tubes before centrifugation; however, in contrast to other methods, ddPCR detected RPP30 in Streck tubes with and without addition of PBS.
Biospecimens
Preservative Types
- Other Preservative
- None (Fresh)
- PAXgene
- Streck/BCT
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer RNA Automated electrophoresis/Bioanalyzer RNA Digital PCR DNA Digital PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Norgen tube
PAXgene tube
Streck tube
Plain tube
Digital PCR Specific Targeted nucleic acid RPP30
Biospecimen Preservation Type of fixation/preservation Blood collection tube additive
None (fresh)
-
Study Purpose
This study compared the fragment size, yield, and purity among cfDNA isolated using different commercial kits from the EDTA plasma of 16 pediatric cancer patients. EDTA plasma from 16 pediatric cancer patients was thawed on ice, aliquoted, and cfDNA was extracted using the QIAamp DNA Blood Mini Kit (QB) (only 7 of 16 patients), QIAamp Circulating Nucleic Acid Kit (QNA), and QIAamp MinElute ccfDNA Mini Kit (QME). EDTA plasma from 29 patients was aliquoted and cfDNA was extracted using the QIAamp MinElute ccfDNA Mini Kit and the NucleoSnap Kit. cfDNA was stored at -80°C. cfDNA (160 bp) and total DNA (50-7000 bp) were quantified by bioanalyzer and cfDNA purity was calculated as the ratio of cfDNA to total DNA. To investigate the quantification methods, cfDNA from culture media and commercial genomic DNA were diluted and quantified using Qubit dsDNA HS Assay Kit, Quant-iT PicoGreen dsDNA Assay Kit, the Bioanalyzer High-Sensitivity DNA Kit, and the TapeStation Cell-free DNA ScreenTape system.
Summary of Findings:
Extraction with the QIAamp DNA Blood Mini Kit resulted in the highest total DNA yield. However, cfDNA yields and purity (ratio of cfDNA to total DNA) were higher when extraction was with the QIAamp Circulating Nucleic Acid Kit or QIAamp MinElute ccfDNA Mini Kit than when extraction was with the QIAamp DNA Blood Mini Kit. Further investigation revealed slightly higher cfDNA yields and lower gDNA contamination when extraction was with the QIAamp MinElute ccfDNA Mini Kit rather than the QIAamp Circulating Nucleic Acid Kit. However, in a follow-up comparison, NucleoSnap yielded more cfDNA from the plasma of 22 or 29 patients than the QIAamp MinElute ccfDNA Mini Kit (P=0.0005). Importantly, the genomic DNA contamination was low using both NucleoSnap and the QIAamp MinElute ccfDNA Mini Kit. Dilution of cell culture cfDNA and commercial genomic DNA showed that Qubit Bioanalyzer DNA HS chip and TapeStation Cell-free DNA tape were generally consistent with expected concentrations but PicoGreen underestimated the cfDNA and genomic DNA concentration. Bioanalyzer analysis showed that size selection using magnetic beads successfully eliminated gDNA while retaining the cfDNA resulting in an increase in the cfDNA fraction from 81% to 95%.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Pediatric
Platform:
Analyte Technology Platform DNA Digital PCR RNA Fluorometry DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method QIAamp DNA Blood Mini Kit
QIAamp Circulating Nucleic Acid Kit
NucleoSnap kit
QIAamp MinElute ccfDNA Mini Kit
Fluorometry Specific Technology platform Qubit dsDNA HS Assay Kit
Bioanalyzer
TapeStation
Analyte Extraction and Purification Analyte purification Size selection with beads
No further purification