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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

PCR amplification of GAPDH and β-actin DNA. DNA was extracted from frozen-liquid samples and dried blood spots. Lanes 1–2 show dsDNA isolated from frozen whole blood and the buffy coat; lanes 3–4, dsDNA isolated from DBSs, with whole blood stored at room temperature; and lanes 5–6, ssDNA isolated from DBSs, with whole blood and the buffy coat stored at room temperature and extracted with the boiling method. BC = buffy coat in dried blood spots stored at room temperature; DBS = dried blood spot; dsDNA = double-stranded DNA; PCR = polymerase chain reaction; ssDNA = single-stranded DNA; WB = whole blood in dried blood spots stored at room temperature.
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fig7: PCR amplification of GAPDH and β-actin DNA. DNA was extracted from frozen-liquid samples and dried blood spots. Lanes 1–2 show dsDNA isolated from frozen whole blood and the buffy coat; lanes 3–4, dsDNA isolated from DBSs, with whole blood stored at room temperature; and lanes 5–6, ssDNA isolated from DBSs, with whole blood and the buffy coat stored at room temperature and extracted with the boiling method. BC = buffy coat in dried blood spots stored at room temperature; DBS = dried blood spot; dsDNA = double-stranded DNA; PCR = polymerase chain reaction; ssDNA = single-stranded DNA; WB = whole blood in dried blood spots stored at room temperature.

Mentions: Nonquantitative PCR amplification of GAPDH and β-actin DNA was performed with (1) ssDNA from frozen-liquid samples, (2) dsDNA from filter paper, and (3) ssDNA from filter paper (Figure 7). The results showed that the DNA obtained from DBS extraction was of sufficient quality to be used for PCR analysis and that the yield was better than that obtained from frozen samples.


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)

PCR amplification of GAPDH and β-actin DNA. DNA was extracted from frozen-liquid samples and dried blood spots. Lanes 1–2 show dsDNA isolated from frozen whole blood and the buffy coat; lanes 3–4, dsDNA isolated from DBSs, with whole blood stored at room temperature; and lanes 5–6, ssDNA isolated from DBSs, with whole blood and the buffy coat stored at room temperature and extracted with the boiling method. BC = buffy coat in dried blood spots stored at room temperature; DBS = dried blood spot; dsDNA = double-stranded DNA; PCR = polymerase chain reaction; 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

fig7: PCR amplification of GAPDH and β-actin DNA. DNA was extracted from frozen-liquid samples and dried blood spots. Lanes 1–2 show dsDNA isolated from frozen whole blood and the buffy coat; lanes 3–4, dsDNA isolated from DBSs, with whole blood stored at room temperature; and lanes 5–6, ssDNA isolated from DBSs, with whole blood and the buffy coat stored at room temperature and extracted with the boiling method. BC = buffy coat in dried blood spots stored at room temperature; DBS = dried blood spot; dsDNA = double-stranded DNA; PCR = polymerase chain reaction; ssDNA = single-stranded DNA; WB = whole blood in dried blood spots stored at room temperature.
Mentions: Nonquantitative PCR amplification of GAPDH and β-actin DNA was performed with (1) ssDNA from frozen-liquid samples, (2) dsDNA from filter paper, and (3) ssDNA from filter paper (Figure 7). The results showed that the DNA obtained from DBS extraction was of sufficient quality to be used for PCR analysis and that the yield was better than that obtained from frozen samples.

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