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Rapid DNA extraction from dried blood spots on filter paper: potential applications in biobanking.

Choi EH, Lee SK, Ihm C, Sohn YH - Osong Public Health Res Perspect (2014)

Bottom Line: A single DBS containing 40 μL blood was used.DNA was efficiently extracted in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer by incubation at 37°C overnight.DNA was stable in DBSs that were stored at room temperature or frozen.

View Article: PubMed Central - PubMed

Affiliation: Eulji Medi-Bio Research Institute (EMBRI), Daejeon, Korea.

ABSTRACT

Objectives: Dried blood spot (DBS) technology is a microsampling alternative to traditional plasma or serum sampling for pharmaco- or toxicokinetic evaluation. DBS technology has been applied to diagnostic screening in drug discovery, nonclinical, and clinical settings. We have developed an improved elution protocol involving boiling of blood spots dried on Whatman filter paper.

Methods: The purpose of this study was to compare the quality, purity, and quantity of DNA isolated from frozen blood samples and DBSs. We optimized a method for extraction and estimation of DNA from blood spots dried on filter paper (3-mm FTA card). A single DBS containing 40 μL blood was used.

Results: DNA was efficiently extracted in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer by incubation at 37°C overnight. DNA was stable in DBSs that were stored at room temperature or frozen. The housekeeping genes GAPDH and beta-actin were used as positive standards for polymerase chain reaction (PCR) validation of general diagnostic screening.

Conclusion: Our simple and convenient DBS storage and extraction methods are suitable for diagnostic screening by using very small volumes of blood collected on filter paper, and can be used in biobanks for blood sample storage.

No MeSH data available.


Related in: MedlinePlus

DNA extracted by boiling dried blood spots on filter paper after incubation at 56°C. BC = buffy coat in dried blood spots stored at room temperature; ssDNA = single-stranded DNA; WB = whole blood in dried blood spots stored at room temperature.
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fig4: DNA extracted by boiling dried blood spots on filter paper after incubation at 56°C. BC = buffy coat in dried blood spots stored at room temperature; ssDNA = single-stranded DNA; WB = whole blood in dried blood spots stored at room temperature.

Mentions: To measure the DNA yields from samples stored under different conditions, samples were separated into whole-blood and buffy-coat samples, spotted on filter papers, and stored at room temperature; subsequently, the extracted ssDNA yields were compared (Figure 4). The amount of ssDNA extracted from buffy-coat samples was approximately 65% higher than that extracted from whole-blood samples after 2 weeks and approximately 44% higher after 4 weeks. Moreover, the purity of ssDNA extracted from buffy-coat samples was higher than that extracted from whole-blood samples (data not shown).


Rapid DNA extraction from dried blood spots on filter paper: potential applications in biobanking.

Choi EH, Lee SK, Ihm C, Sohn YH - Osong Public Health Res Perspect (2014)

DNA extracted by boiling dried blood spots on filter paper after incubation at 56°C. BC = buffy coat in dried blood spots stored at room temperature; ssDNA = single-stranded DNA; WB = whole blood in dried blood spots stored at room temperature.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4281615&req=5

fig4: DNA extracted by boiling dried blood spots on filter paper after incubation at 56°C. BC = buffy coat in dried blood spots stored at room temperature; ssDNA = single-stranded DNA; WB = whole blood in dried blood spots stored at room temperature.
Mentions: To measure the DNA yields from samples stored under different conditions, samples were separated into whole-blood and buffy-coat samples, spotted on filter papers, and stored at room temperature; subsequently, the extracted ssDNA yields were compared (Figure 4). The amount of ssDNA extracted from buffy-coat samples was approximately 65% higher than that extracted from whole-blood samples after 2 weeks and approximately 44% higher after 4 weeks. Moreover, the purity of ssDNA extracted from buffy-coat samples was higher than that extracted from whole-blood samples (data not shown).

Bottom Line: A single DBS containing 40 μL blood was used.DNA was efficiently extracted in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer by incubation at 37°C overnight.DNA was stable in DBSs that were stored at room temperature or frozen.

View Article: PubMed Central - PubMed

Affiliation: Eulji Medi-Bio Research Institute (EMBRI), Daejeon, Korea.

ABSTRACT

Objectives: Dried blood spot (DBS) technology is a microsampling alternative to traditional plasma or serum sampling for pharmaco- or toxicokinetic evaluation. DBS technology has been applied to diagnostic screening in drug discovery, nonclinical, and clinical settings. We have developed an improved elution protocol involving boiling of blood spots dried on Whatman filter paper.

Methods: The purpose of this study was to compare the quality, purity, and quantity of DNA isolated from frozen blood samples and DBSs. We optimized a method for extraction and estimation of DNA from blood spots dried on filter paper (3-mm FTA card). A single DBS containing 40 μL blood was used.

Results: DNA was efficiently extracted in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer by incubation at 37°C overnight. DNA was stable in DBSs that were stored at room temperature or frozen. The housekeeping genes GAPDH and beta-actin were used as positive standards for polymerase chain reaction (PCR) validation of general diagnostic screening.

Conclusion: Our simple and convenient DBS storage and extraction methods are suitable for diagnostic screening by using very small volumes of blood collected on filter paper, and can be used in biobanks for blood sample storage.

No MeSH data available.


Related in: MedlinePlus