Improving DNA extraction quality by the salting‐out method to prepare blood samples for real‐time PCR. (LB124)

PCR, or “polymerase chain reaction”, is one of most useful techniques in genomics research. High quality DNA extraction is necessary to obtain satisfactory results. Some methods utilized to obtain DNA from blood include a simple salting‐out procedure for extracting DNA from human nucleated cells. This method is widely used by many laboratories because it can obtain DNA with sufficient quality to realize PCR, using materials that do not pose risk to researchers and is more cost‐efficient than other approaches. Although salting‐out has spread to many laboratories, its protocols do not highlight technical details of their steps that could affect final DNA quality and, for this reason, underwent some modifications in this work in order to obtain an efficient extraction. We used 2 to 4 mL of blood for each of 30 DNA samples. For the supernatant step in first EDTA wash, we established a subtraction pattern from 15 mL to 2.5 mL, independently of initial sample volume of blood, to avoid losing leucocytes. The number of washes required is directly proportional to loss of leukocytes in the blood sample, considering that 4 mL samples can receive five washes while 2 mL samples tolerate only four. It was noted that a “centrifuge brake” can reduce DNA extraction quality, thus indicating its inadvisability. All DNA samples were submitted to spectrophotometry, which showed the DNA/Protein ratio staying between 1.1 and 1.6. According to the literature, 93% of our samples were ready for real‐time PCR after modifications, indicating that those modifications were relevant for achieving efficient extraction. Integrity was confirmed by electrophoresis. All modifications made in the salting‐out method have improved the quality and integrity of DNA samples. Grant Funding Source : FAPESP 2013/19808‐8