When considering methods to extract nucleic acids (DNA and RNA) to be used in downstream applications, it is important to think about what the sample source will be, as well as if it will be able to be used in an eventual assay. Being able to extract and purify DNA can lead to important information on infectious disease testing as well as patient genotype analysis. Purification of RNA can aid in understanding gene expression. However, once nucleic acid is extracted from a specimen, an additional set of parameters and guidelines are put in place to ensure that quality standards are met and the nucleic acid has not been compromised, ultimately leading to its ability to be used in patient testing.
Specimens / Sample Sources
Fresh Tissue in the form of cells such as, buccal cells, swabbed cervical cells, and even fresh DNA from the root of a single hair are all easily collected, stored, and provide enough DNA to be used in downstream PCR assays.
Whole Blood samples are easily available and require relatively simple methods to extract nucleic acid. However, the main source of issues comes from the type of anticoagulant that the blood specimen is mixed with. EDTA, Heparin, and ACD are all available as coating in blood collection tubes and are used to prevent clotting of the sample. Studies have shown that EDTA and ACD are acceptable anticoagulants that minimally interfere the quality and quantity of extracted nucleic acid as well as downstream PCR applications. Conversely, heparin is not completely removed during extraction procedures and can have an inhibitory effect on PCR and other enzyme based assays. Methods to eliminate heparin from a sample can be employed and include ethanol precipitation, boiling, and filtering.
Formalin-Fixed, Paraffin-Embedded Tissues provide a wealth of samples as these types of preserved tissues are typically stored in great numbers by pathology departments. The problem with tissues preserved in this manner is that quality is definitely compromised. When nucleic acids are exposed to fixatives, such as formalin, they become seriously fragmented and degraded. While these sources of samples aren’t ideal, there still are many benefits to using formalin-fixed or paraffin-embedded samples.
While most high volume laboratories employ automated extraction methods carried out on large pieces of instrumentation, there are laboratories that perform manual extractions at the bench. Manual extraction provides some of the best quality of nucleic acid samples. Typically, the purity of the nucleic acid is slightly compromised when automated methodology is employed; however the effect is somewhat negligible and allows a high volume of samples to be processed in various downstream applications.
Regardless of the sample type, the first steps of any extraction involve tissue isolation, disruption, and lysis of the cells. Paraffin-embedded samples require an initial step to remove the paraffin from the sample and this is accomplished via heat or the use of xylene. When isolating nucleic acids from blood, the white blood cells are isolated prior to extraction. Molecular detergents, such as SDS (Sodium Dodecyl Sulfate) are utilized to lyse the cells. Sometimes laboratories include a Proteinase K step due to the high amount of protein present in cell lysates.
Extraction methods vary, but the most common fall into three typical groups: Organic, Inorganic, or Solid Phase. Organic extractions utilize organic chemicals such as phenol or chloroform. Inorganic extractions use inorganic chemicals such as detergents, EDTA, acetic acid, or salt. Solid phase extractions immobilize nucleic acid on solid support system such as a spin columns or beads. Many automated extraction instruments employ silica bead based extraction chemistry.
Quality Assessment of DNA (Measurements)
Many PCR assays require a quality check for the nucleic acid purity and/or concentration. Spectrophotometers are an easy way to assess these two quality measures. Nucleic acids exhibit maximum absorption at 260nm and proteins at 280nm. Therefore, purity of a sample is assessed by measuring the optical density (OD) at 260nm and 280nm and calculating the ratio: OD260/280. A nucleic acid purity ratio of 1.8 – 2.0 is considered relatively pure. A reading less than 1.8 suggests protein contamination and readings above 2.0 suggest increased presence of RNA.
Note: An OD260 of 1.0 corresponds to 50 μg/mL of double stranded DNA or 40 μg/mL of RNA
A simple formula is used to calculate the quantity (concentration) of nucleic acid:
Where: A = Absorbance
ε = molar absorptivity
DNA is 50 L/mol-cm and RNA is 40 L/mol-cm
b = Path length (cm)
c = Concentration (mg/L)
Storage of Nucleic Acid
DNA can safely be stored long term if stored in Tris-EDTA buffer at 4°C. Typically, the colder the temperature, the less chance for degradation. Ideally, store DNA at -80°C and reduce the amount of freeze-thaw cycles. RNA should be stored in the same type of buffer at -80°C.
Test your Knowledge!
- The following DNA samples are extracted from a whole blood sample and assessed on a spectrophotometer. Calculate the purity of each sample and comment on its quality:
- Calculate the concentration of DNA and RNA if the A260 reading = 0.225 at a 1:100 dilution and the spectrophotometer has a 1.0 cm pathlength.
Sample 1 = 1.85. The purity is considered ideal for DNA
Sample 2 = 1.00 which suggests protein contamination
Sample 3 = 2.11 which is ok for RNA
2. Solving for concentration (“c” from the formula above)
DNA: 1125 μg/mL
RNA: 900 μg/mL
-LeAnne Noll, BS, MB(ASCP)CM is a molecular technologist at Children’s Hospital of Wisconsin and was recognized as one of ASCP’s Top Five from the 40 Under Forty Program in 2015.