Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Method Validation for Extraction of Nucleic Acids from Peripheral Whole Blood.

Author(s): Mathay C, Hamot G, Henry E, Mommaerts K, Thorlaksdottir A, Trouet J, Betsou F

Publication: Biopreserv Biobank, 2016, Vol. 14, Page 520-529

PubMed ID: 27548617 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study sought to optimize the extraction of DNA from blood and buffy coat specimens by comparing DNA yield and purity following extraction from different volumes of blood (2, 3, and 4 mL), using manual versus automated lysis, using different elution volumes (300 or 600 µL), and following storage of lysate (not stored versus 2- 8°C  for 1 week) and storage of extracted DNA(in 13 and 1.5 mL tubes on Chemagenic racks or 1.5 mL tubes not on racks).  Blood was collected from three or more volunteers (no diagnosis) into K₂EDTA vacutainer tubes and centrifuged at 2,000 x g for 20 min at 18-22°C. Buffy coat was automatically removed using a Freedom EVO liquid handler and then 1 mL aliquots were transferred to conical tubes and diluted with 4 mL water.  DNA was extracted from blood and buffy coat using the Chemagic DNA Blood Kit Kit and the automated Chemagic MSM I protocol. Extracted DNA was stored at -80°C. DNA yield and A260/A280 were assessed by spectrophotometer. To Potential effects of the volume of blood used for DNA extraction was investigated using specimens collected from three healthy volunteers; DNA was extracted from 2, 3, and 4 mL aliquots of blood diluted with water to obtain a final volume of 4 mL. To investigate the Potential effects of manual and automated lysis were investigated using the blood and isolated buffy coat of two volunteers; buffy coat specimens were lysed manually with a vortex, digested with proteinase K and used immediately for extraction while matched aliquots were treated with lysis buffer with proteinase K using the non-prefilled program on the MSM I instrument and then stored at room temperature for 2 h before extraction. Potential effects of elution volume were investigated using blood collected from two volunteers; blood specimens were manually lysed and DNA was extracted using a MSM1 instrument and eluted with either 200 or 600 µL of elution buffer. Reproducibility was assessed using blood aliquots from a single donor; blood aliquots were lysed and stored at 2-8°C before extraction on three consecutive days.  Robustness of the DNA extraction method was investigated by extracting DNA from matched blood and buffy coat from a single volunteer immediately or after lysate was stored for 1 week at 2-8°C.  The robustness of storing extracted DNA on a Chemagen magnetic rack was investigated using blood collected from two volunteers; DNA extracted from buffy coat stored in 13 mL and 1.5 mL tubes in Chemagen magnetic racks compared to matched DNA samples stored in 1.5 mL tubes that were not placed on the rack.

   Summary of Findings:

   DNA yield and purity (A260/A280) were not significantly different (based on acceptance criteria) when DNA was extracted from 2 or 3 mL of blood instead of 4 mL; further,  the DNA yield from 2 and 3 mL of blood was greater than the acceptable threshold for 4 mL of blood (>40% from 2 mL and >70% from 3 mL). DNA yield and purity also met the acceptance criteria (no significant difference in yield or A260/A280 ratio) regardless of the elution volume (300 or 600 µL), or lysis method (manual versus automated).  Consequently, the authors opted to use 4 mL of blood, manual lysis, and a 300 µL elution volume to investigate reproducibility. The coefficient of variance (CV) of DNA yield and purity were <10% in replicate DNA extractions from the same lysate conducted on three consecutive days. The method was considered robust, as the yield and purity of DNA was acceptable (>15 µg DNA, A260/A80 >1.8, and CV <10%) regardless of whether blood buffy coat was used for extraction, whether the lysate was or stored for 1 week at 2- 8°C prior to extraction or extraction was immediate, and whether isolated DNA was stored at -80°C with or without a magnetic rack.

    

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Buffy Coat

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Buffy coat Whole blood
   Biospecimen Aliquots and Components	Aliquot size/volume	2 mL 3 mL 4 mL
   Analyte Extraction and Purification	Analyte isolation method	Manual lysis Automated lysis
   Storage	Storage duration	0 h 1 week
   Storage	Storage conditions	Extracted DNA on magnetic rack in 1.5 mL tube Extracted DNA on magnetic rack in 13 mL tube Extracted DNA not on magnetic rack in 1.5 mL tube
   Analyte Extraction and Purification	Rehydration of dried sample/specimen	300 µL 600 µL

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2. Study Purpose

   This study sought to optimize the extraction of RNA from PAXgene preserved blood by comparing RNA yield, purity and integrity following elution with different buffers, blood storage durations, and extraction using multiple automated instruments.  Levels of miR-16 and RNU24 were also compared following extraction with the different instruments. Blood was collected from six volunteers (no diagnosis specified) into PAXgene tubes and stored at room temperature for 2 h. RNA was extracted immediately or after storage at -20°C for 24 h followed by storage at -80°C (duration not specified). RNA was extracted using the PAXgene Blood miRNA Kit and a QiaCube instrument. Isolated RNA was analyzed immediately and after storage at -80°C. RNA yield and purity (A260/A180 nm) were assessed using a spectrophotometer and integrity was evaluated using the RNA 6000 NanoChip on an Agilent Bioanalyzer 2100. Levels of twomiRNAs (RNU24 and miR-16) were quantified using TaqMan real-time RT-PCR. The acceptance criteria for reproducibility was a CV <30%. Potential effects of elution buffer was investigated using the blood of a single volunteer; extracted RNA was eluted in water, tris EDTA, or BR5 elution buffer. To investigate the Potential effects of PAXgene blood storage was evaluated using the blood of a single volunteer; matched tubes of PAXgene blood were stored for 2 h at room temperature, 18-24 h at room temperature, 2 h at room temperature followed by 1-2 weeks at 2–8°C, or for 2 h at room temperature followed by 1 week at -20°C. Potential effects of RNA extraction method were explored using blood collected from two subsets of three volunteers; RNA was isolated from PAXgene blood (stored frozen at -20°C for 24 h followed by -80℃ and shipment on dry ice) using the QiaCube, Biorobot Universal and QiaSymphony protocols/instruments (3 volunteers) and the MSM I, Biorobot Universal and QiaSymphony protocols/instruments (3 volunteers). To compare the QiaCube and Biorobot instruments, RNA was extracted from ten tubes of PAXgene blood (same blood specimen) using each protocol/instrument. Additionally, extraction was performed on ten specimens that were “difficult to lyse” using the Qiacube instrument and 96 other specimens using the Biorobot instrument.

   Summary of Findings:

   The CV for RNA yield was <30% when PAXgene blood specimens from three patients were analyzed in triplicate. There were no statistically significant differences between elution in water, tris EDTA or BR5 elution buffer, but the CV for yield and RIN were non-significantly lower when BR5 was used as the elution buffer (8.7% and 0.8%, respectively versus 19.6% and 5.5% for water and 19.4% and 1.6% for tris EDTA). Significant effects associated with storing blood in PAXgene tubes were observed (P=0.038 and P=0.011, respectively), with lower RNA yield when specimens were stored for 1 week at -20°C and increased RIN when they were stored at 2–8°C for 2 weeks or at -20°C for 1 week compared to blood stored in PAXgene tubes for 18-24 h at room temperature (P<0.05, all). Isolation of RNA with the MSM instrument resulted in low purity (A260/A280 =1.52), while RNA extracted with Biorobot had a low mean RIN (6.9). All other instruments yielded RNA of acceptable purity (A260/A280 >1.8) and a mean RIN >7. The mean RIN was significantly higher when RNA extraction was with a MSM1 (9.4) or QIAcube (9.1) instrument compared to extraction with a biorobot (P<0.002).  Spectrophotometric RNA yields were higher using the QIAsymphony than the Biorobot instrument (P<0.002). Levels of miR-16 were highest when the Biorobot, QiaCube or QiaSymphony instruments were used and were significantly lower when the MSM instrument was used compared to when the QiaSymphony was used for extraction (P<0.001). Levels of RNU24 were higher when RNA extraction was with QiaSymphony instrument compared to either the MSM or Biorobot instrument (P=0.002). Changing of protocol used with the Biorobot (from default to “No clot detection”) led to higher mean RNA yields (6.57 µg) and purity (A260/A280 =1.95), the yields were still slightly higher using the QiaCube instrument than the Biorobot (8.18µg versus 6.29µg). In specimens that were considered “difficult to lyse”, mean yields were comparable when RNA extraction was with the QiaCube or Biorobot instrument (6.97 µg versus 6.26 µg), and mean RIN and purity were acceptable when extraction was with either the QiaCube (8.1 and 2.25, respectively) or the Biorobot instrument (7.5 and 1.97, respectively).  

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • PAXgene

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	18-24 h at room temperature 2 h at room temperature followed by 1-2 weeks at 2–8°C 2 h at room temperature followed by 1 weeks at -20°C
   Analyte Extraction and Purification	Analyte isolation method	QiaCube Biorobot Universal QiaSymphony MSM I
   Analyte Extraction and Purification	Rehydration of dried sample/specimen	Water Tris EDTA BR5 elution buffer

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