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Tutorials : Sep 15, 2011 ( )
Assuring Reliability of qPCR & RT-PCR Results
Use of Spectrophotometry on Nucleic Acid Samples Before Experiment Improves Outcome
The polymerase chain reaction (PCR) is a valuable tool used in both research and molecular diagnostic laboratories because of its specificity, efficiency, fidelity, and relative ease of use.
Quantitative real-time PCR (qPCR) enables sensitive and accurate quantitative measurement of nucleic acids. Both qPCR and reverse transcriptase PCR (RT-qPCR) are used across a wide range of applications such as gene expression, SNP genotyping, copy-number analysis, pathogen detection, drug target validation, and measurement of RNA interference (RNAi).
The quality of qPCR and RT-qPCR results can be negatively affected by many experimental variables. To ensure the validity of assay results, sample extraction and preparation steps must be closely monitored, and the starting material must be well characterized before performing RT and qPCR assays.
Slight differences in pipetting, lack of instrument calibration, improper choice of reference genes, incorrect quantification, and/or use of impure nucleic acid templates can generate erroneous, but believable, results. Therefore, the use of standardized best practices to ensure reliable and meaningful results is recommended. To address the need for standardized qPCR practices, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines have been developed.
The MIQE Guidelines
The MIQE guidelines define the minimum information necessary for proper evaluation of qPCR experiments and publications. By detailing all relevant experimental conditions and assay characteristics, the validity of the protocols used can be thoroughly assessed. Complete disclosure of all reagents, sequences, and analysis methods used enables other investigators to reproduce results.
The guidelines address several general categories including sample preparation, QC of nucleic acids, RT, qPCR assay, and data analysis. They also include an extensive checklist of “essential” or “desirable” items to be included in each of the following sections: experimental design, sample, nucleic acid extraction, RT, target information, primers and probe, qPCR protocol, qPCR validation and data analysis.
Most of the MIQE guidelines can be adopted quickly. Adoption of the “essential” items ensures that key variables affecting data quality are addressed, which, in turn, increases the degree of confidence in the results and the conclusions drawn. Adoption of some or all of the “desirable” items further ensures that a comprehensive approach is being taken to obtain quality data.
For instance, the guidelines recommend the disclosure of the probe sequence as highly desirable and strongly encouraged, but not all vendors of predesigned assays provide this information. Thermo Fisher Scientific provides probe sequences for all Solaris qPCR gene-expression assays.
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.
Assuring Purity through Spectral Analysis
A260/A280 and A260/A230 ratios are routinely used for purity assessment of nucleic acids. The A260/A280 ratio, which is an indication of the possible presence of protein, should be approximately 1.8 for pure DNA and 2.0 for pure RNA. Nucleic acids should have an A260/A230 ratio between 1.8 and 2.2.
Because impure samples can sometimes have ratios within the acceptable ranges, researchers should also examine the spectral data. This data can provide additional information regarding potential chemical contaminants (e.g., phenol and guanidine) that may be introduced by extraction procedures and can act as PCR inhibitors.
Because many chemical contaminants exhibit characteristic spectral profiles, spectral analysis can be used to identify the source of contamination. For example, a sample containing phenol will have increased absorbance both at 270 nm and below 240 nm. These changes in sample absorbance lower the A260/A230 ratio and shift the spectral peak to about 270 nm (Figure2).
In contrast, a DNA sample containing guanidine hydrochloride and a sample of pure DNA will have similar spectral profiles in the 260 nm to 280 nm region. However, the presence of guanidine increases the absorbance in the spectral region below 240 nm and therefore, lowers the A260/A230 ratio (Figure 2).
The broad utility of qPCR and RT-qPCR has brought forth a myriad of applications across a wide range of scientific and clinical disciplines. However, a lack of consensus existed on how to best perform and evaluate qPCR experiments. The MIQE guidelines have been developed to encourage best practices and allow reliable and unequivocal interpretation of qPCR results.
Best practices in qPCR first focus on the pre-qPCR steps, because inaccurate characterization of the starting material may generate unreliable qPCR results. The NanoDrop 2000 series can be used to determine the concentration and purity of nucleic acid samples before a qPCR experiment.
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