Accurate Template Qualification as a Quality Control Measure
The MIQE guidelines recommend quantification and quality assessment of nucleic acids. Thermo Scientific NanoDrop spectrophotometers provide reliable determination of nucleic acid concentration and purity. When performing absolute quantification of target sequences with qPCR, prior knowledge of sample concentration is important, because sample values must fall within the standard curve.
Precise quantification of unknown nucleic acid samples by spectrophotometry ensures that the resulting Cq values lie within the linear portion of the standard curve and that accurate and reproducible data are obtained.
Relative quantification is suitable for measuring changes in gene expression under various conditions. Variability in gene-expression assays is frequently caused by the use of very low template quantities. Relative quantification can also be used for SNP genotyping.
When performing this type of assay, it is important for the signal strength in the unknown and control samples to be similar. SNP genotyping is also highly dependent on template concentration. Low template quantities make allele calls difficult or unreliable (Figures 1A+B).
Validation of new reference genes requires accurate quantification of input RNA to ensure that the proper reference genes are chosen. Moreover, accurate quantification of nucleic acids is essential when appropriate reference genes cannot be identified and gene-expression studies are normalized to total RNA input.
Nucleic acid concentrations have been traditionally measured by using a spectrophotometer to determine the absorbance of UV light at 260 nm (A260). Typically, quantification of nucleic acids is performed after nucleic acid extraction.
The NanoDrop 2000 series UV-Vis spectrophotometer facilitates rapid measurements of very small sample volumes (0.5 µL—2.0 µL), with a dynamic range of 2 ng/µL—15,000 ng/µL for DNA, without the need for a cuvette or dilutions. The 2000c cuvette capability extends the detection limit down to 0.4 ng/μL by extending the pathlength.
The spectrophotometer determines nucleic acid concentration and generates full absorbance spectral data. Both RNA and DNA absorb UV light at 260 nm; therefore, an absorbance measurement determines total nucleic acid concentration.