Influence of plasma processing on recovery and analysis of circulating nucleic acids.

Author(s): Page K, Guttery DS, Zahra N, Primrose L, Elshaw SR, Pringle JH, Blighe K, Marchese SD, Hills A, Woodley L, Stebbing J, Coombes RC, Shaw JA

Publication: PLoS One, 2013, Vol. 8, Page e77963

PubMed ID: 24205045 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effect of cfDNA extraction method and centrifugation speed on the yield of amplifiable DNA and deep sequencing results in plasma. The effect of DNA extraction method on cfDNA yield was determined using pooled plasma from 3 healthy donors. To determine the effect of extraction method on deep sequencing results, venous blood from 4 patients with metastatic breast cancer was drawn into EDTA tubes and centrifuged at 1000 x g for 10 min at 4 degrees C, and plasma was removed. Plasma was centrifuged at 1000 x g (or 2000 x g, or 10000 x g) for 10 min and stored at -80 degrees C.

   Summary of Findings:

   There was no effect of the speed of the second centrifugation step on the CT of GAPDH. cfDNA yield, as determined by real-time PCR, was highest when cfDNA was extracted from the single plasma pool using the QIAamp DNA Blood Mini or CNA kits, with 1/10 as much cfDNA obtained using the NucleoSpin Plasma XS Kit or the FitAmp Plasma/Serum DNA Isolation kit. Recovery of a 23 kb fragment of spiked DNA was comparable between the two QIAamp kits, but recovery of a 564 bp fragment was highest using the QIAamp CNA kit. Overall, the number of deep sequencing reads for cfDNA isolated using the two QIAgen kits was very strongly correlated (r>0.95), but in one specimen, the correlation was strong (r=0.87) with more uniform coverage observed using cfDNA isolated with the QIAamp CNA kit. Of the 167 sequence variants identified, 136 were identified at a similar rate using cfDNA isolated with either kit, and 31 were identified using only one kit, and these generally were detected with fewer reads.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Next generation sequencing

   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 kits NucleoSpin Plasma XS Kit FitAmp Plasma/Serum DNA Isolation kit
   Real-time qPCR Specific	Targeted nucleic acid	HPRT1 GAPDH Beta actin
   Biospecimen Aliquots and Components	Centrifugation	Multiple speeds compared

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2. Study Purpose

   The purpose of this study was to determine the effects of delayed centrifugation of blood, centrifugation speed, extraction method, storage duration and array platform on miRNA yield and quantification in plasma. To determine the effects of extraction method, pooled purchased plasma and plasma specimens from 3 patients with primary or metastatic breast cancer were used. To investigate the effects of plasma processing, blood specimens were obtained from 5 healthy patients. To determine the effects of storage, two plasma pools consisting of 10 specimens each from patients with metastatic breast cancer were used. Venous blood was drawn into EDTA tubes, split into two specimens and centrifuged 2 h or 6 h later at 1000 x g for 10 min at 4 degrees C, and plasma was removed. Plasma was recentrifuged at different speeds (1000 x g, 2000 x g, or 10000 x g) and stored at -80 degrees C. After thawing, plasma was centrifuged at 1000 x g for 5 min at room temperature and placed in a new tube before DNA or miRNA extraction.

   Summary of Findings:

   The authors report it was not possible to obtain sufficient miRNA to amplify miR-21 or miR-191 from 1 mL of purchased pooled plasma. When plasma was pooled from 3 patients with primary breast cancer, significantly more miRNA was obtained using the QIAamp CNA or miRNeasy Plasma/Serum kit than the miRvana microRNA Isolation kit (p <0.001). The cycle threshold values for miR-21 and miR-191 were lower when miRNA was isolated using the miRNeasy Serum/Plasma kit than the QIAamp CNA and miRvana kits. However, the miRNA profile, as determined by low density array was comparable between different extraction methods, and the miRNeasy Serum/Plasma kit was chosen for the remaining work. miRNA yield was not affected by the volume of plasma used or delayed centrifugation, but levels of miR-15b (p<0.001), miR-16 (p<0.05), miR-21 (p<0.01), miR-24 (p<0.01) and miR-484 (p<0.05) were significantly reduced, and levels of miR-191 (p<0.001) were increased when centrifugation was delayed. When the second centrifugation speed was increased from 1000 x g to 2000 x g, the number of miRNAs detected decreased from 195 to 187, and when the speed was further increased to 10,000 x g, only 138 miRNAs were detected. While the relative CT values of most miRNAs were unaffected by the centrifugation speed, the platelet associated miRNAs were selectively reduced by faster centrifugation. While a very strong correlation in miRNA levels was observed for replicate specimens using both low density array platforms (r=0.95), there was only a modest correlation between miRNA levels in the two platforms (r=0.60). Further, non-specific amplification, as determined by melting curve analysis, was observed for the low abundance miRNAs using the Exiqon miRCURY LNA miRNA PCR array. Pooled plasma stored for >12 years at -80 degrees C only allowed for detection of 177 miRNAs, but 202 were detected in freshly frozen plasma. However, the CT values were comparable between stored and unstored plasma.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Low density array
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	2 h 6 h
   Storage	Storage duration	0 years >12 years
   Analyte Extraction and Purification	Analyte isolation method	miRNeasy Serum/Plasma Kit miRvana microRNA Isolation kit QIAamp Circulating Nucleic Acid kit
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-21 miR-191
   Biospecimen Aliquots and Components	Aliquot size/volume	200 uL 500 uL 1 mL
   Low density array Specific	Type of array	Exiqon miRCURY LNA microRNA PCR array TLDA
   Biospecimen Aliquots and Components	Centrifugation	Multiple speeds compared Centrifugation delays investigated

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