Direct comparison of QIAamp DSP Virus Kit and QIAamp Circulating Nucleic Acid Kit regarding cell-free fetal DNA isolation from maternal peripheral blood.
Author(s): Jain M, Balatsky AV, Revina DB, Samokhodskaya LM
Publication: Mol Cell Probes, 2019, Vol. 43, Page 13-19
PubMed ID: 30584912 PubMed Review Paper? No
Purpose of Paper
This paper compared the yield of cell-free fetal DNA (cffDNA) and total cell-free DNA (cfDNA) and PCR inhibition between DNA extracted from plasma using the QIAamp Circulating Nucleic Acid (CNA) and QIAamp DSP virus (DSP) kits. The effect of using a lower plasma volume in conjunction with the DSP kit was also investigated.
Conclusion of Paper
The yield of cffDNA and total cfDNA were significantly higher using the CNA kit than the DSP kit, when an equivalent input volume was used (1 mL). cfDNA extracted with each kit displayed PCR inhibition, but a lower amount of cfDNA extracted using DSP than CNA was required to cause inhibition. When the recommended input volume of 0.5 mL plasma was used with the DSP kit the cfDNA yield increased significantly, indicating that the DSP kit was overloaded by the initial input volume.
Studies
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Study Purpose
This study compared the yield of cffDNA and cfDNA and PCR inhibition between DNA extracted from plasma using the QIAamp CNA and DSP kits. The effect of using a lower plasma volume in conjunction with the DSP kit was also investigated. Blood was collected from 18 women in the 6-14th week of pregnancy and from 12 healthy non-pregnant women into EDTA tubes and immediately placed at 4˚C. Within 4 h, plasma was obtained by dual centrifugation at 3000 x g and frozen at -80˚C. Specimens were thawed and split into 1 mL aliquots for extraction with the QIAamp DSP Virus Kit and QIAamp Circulating Nucleic Acid Kits. Carrier RNA was used in conjunction with each protocol. Isolated cfDNA was stored at -80˚C. An aliquot of DNA was restriction-digested with the methylation-sensitive enzyme BstFN1 and confirmed by the lack of real-time PCR amplification of unmethylated ACTB. cffDNA and cfDNA were quantified by dPCR amplification of RASSF1A. To test for the presence of PCR inhibitors, amplification of the Epstein Barr Virus (EBNA-1) in the presence of increasing concentrations of extracted cfDNA was investigated.
Summary of Findings:
Almost 3-fold more cffDNA was obtained using the CNA kit than the DSP kit (167.62 versus 52.88 GEq/mL, P<0.001). Similarly, the total cfDNA yield was approximately 2-fold higher using the CNA kit than the DSP kit (743.42 versus 371.07 GEq/mL, P<0.001). Although the percentage of fetal DNA isolated was higher using CNA than DSP (24.75% versus 14.20%), the difference was not significant (P=0.0586). Inhibition of EBNA-1 amplification was observed with addition of 5 or 10% cfDNA isolated with the DSP kit (P=0.002 and P=0.041, respectively) and 10% of cfDNA isolated with the CNA kit (P=0.002), but the inhibition was greater when 5% DNA isolated with DSP was added than when 10% was added. Importantly, the yield of total cfDNA was 1.8-fold higher (1,799.025 versus 1,041.061 GEq/mL P=0.028) when 0.5 mL plasma was used as input for the DSP kit instead of 1 mL. Thus the authors state that the DSP kit should be used only with the recommended 0.5 mL input.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Pregnant
Platform:
Analyte Technology Platform DNA Digital PCR DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method QIAamp DSP Virus Kit
QIAamp Circulating Nucleic Acid Kit
Biospecimen Aliquots and Components Aliquot size/volume 0.5 mL
1.0 mL