Upstream processing or contaminating nucleases can lead to degradation or fragmentation, and problems with enzymatic reactions and biochemical modifications may result in unexpected fragment sizes. Therefore, it is important to check the condition of your DNA or RNA samples prior to analysis.
These issues can be easily checked by analyzing the size distribution pattern of the samples. Along with quantity and purity, size distribution is a critical QC parameter that provides valuable insight about sample quality. Analyzing nucleic acid size distribution through manual observation or using dedicated algorithms informs you about your sample’s integrity and indicates whether the samples are fragmented or contaminated by other DNA or RNA products.
Back to topInsight offered by nucleic acid size distribution
Checking the size distribution of your sample by various electrophoretic methods can provide valuable insights into the outcome and success of many different procedures.
Analyzing PCR amplicons or RFLP fragments confirms the presence of the expected size fragments and alerts you to the presence of any non-specific amplicons. Electrophoresis also helps you assess the ligation efficiency yield for plasmid cloning procedures as well as the efficiency of removal of primer–dimers or other unspecific fragments during sample cleanup.
For complex samples such as genomic DNA (gDNA) or total RNA, the shape and position of the smear from electrophoresis analysis directly correlates with the integrity of the samples. Nucleic acid species of larger size tend to be degraded first and provide degradation products of lower molecular weight. Samples of poor integrity generally have a higher abundance of shorter fragments, while high-quality samples contain intact nucleic acid molecules with higher molecular size.
Eukaryotic RNA samples have unique electrophoretic signatures, which consist of a smear with major fragments corresponding to 28S, 18S and 5S ribosomal RNA (rRNA). These electrophoretic patterns correlate with the integrity of the RNA samples. The RNA integrity can either be assessed manually or with automation that employs a dedicated algorithm such as the RNA Integrity Score (RIS) that gives an objective integrity grade to RNA samples ranging from 1–10. RNA samples of highest quality usually have a score of 8 or above.
For preparation of NGS libraries, checking the sample size distribution after target enrichment, shearing, adapter ligation and final size selection ensures that the process will provide high-quality libraries and high-quality sequencing data.
Back to topRelevance and impact on downstream analysis steps
A sample’s size distribution directly reflects its integrity and suitability for use in downstream applications such as genotyping, gene expression or sequencing. Low-quality nucleic acid samples with high levels of degradation can negatively affect PCR reactions and lead to errors in replication, lower amplification yields and incorrect CT
values. In the worst cases, fragile PCR or sequencing targets can be completely lost or degraded during upstream workflow steps. This increases the risk of false-negative results and invalidation of quantitative assays.
Quality control of NGS libraries through size distribution analysis lets you know whether all of the library preparation steps were successful. It also lets you know if the size selection process has correctly removed unwanted fragments and primer/adaptor–dimers. In addition, it ensures that the average size range of fragments in the library is compatible with the sequencer’s read length capacity. Using libraries of incorrect size can lead to loss in sequencing coverage for some genetic regions.
Back to topTechnologies for assessing nucleic acid size distribution
Electrophoretic separation of nucleic acids is the most commonly used lab technique for assessing a nucleic acid sample’s size distribution. Negatively charged nucleic acid molecules migrate through a matrix towards the anode, separating and resolving the nucleic acids according to their size.
Modern and high-performance technologies such as capillary electrophoresis (CE) have replaced agarose gel electrophoresis for many situations, providing highly resolving and sensitive nucleic acid analysis that is faster and safer.
In CE, charged DNA or RNA molecules are injected into a capillary and are resolved during migration through a gel-like matrix. Nucleic acids are detected as they pass by a detector that captures signals of specific absorbances. Results are presented in the form of electropherogram, which is a plot of signal intensity against migration time. The fragment sizes are precisely determined using a size marker consisting of fragments of known size.
Back to topQIAGEN solutions
The QIAxcel Advanced system
replaces traditional, labor-intensive DNA and RNA gel analysis, streamlining workflows and reducing time to result. QIAxcel Advanced fully automates sensitive, high-resolution capillary electrophoresis of up to 96 samples per run. DNA fragment analysis of 12 samples can be performed in as little as 3 minutes. Ready-to-run gel cartridges allow 96 samples to be analyzed with a minimum of hands-on interaction, reducing manual handling errors and eliminating the need for tedious gel preparation. User-friendly QIAxcel ScreenGel software ensures convenient analysis and documentation of data. The QIAGEN RNA Integrity Score (RIS) provides an objective quality measurement of the analyzed samples and allows easy interpretation of sample integrity.
Find out more about other important parameters for sample QC:
Back to topReferences
- Unger, C., Kofanova, O., Sokolowska, K., Lehmann, D. and Betsou, F. (2015) Ultraviolet C radiation influences the robustness of RNA integrity measurement. Electrophoresis, 36, 2072–2081. doi:10.1002/elps.201500082
- Cuthbertson, L. et al. (2015) Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses. J. Cyst. Fibros. 14:4, 464–467.
- Sidova, M., Tomankova, S., Abaffy, P., Kubista, M., and Sindelka, R. (2015) Effects of post-mortem and physical degradation on RNA integrity and quality. Biomolec. Det. and Quant. 5,3–9.
- Groelz, D., Sobin, L., Branton, P., Compton, C., Wyrich, R., and Rainen, L. (2013) Non-formalin fixative versus formalin-fixed tissue: A comparison of histology and RNA quality. Exp. Molec. Path. 94:1, 188–194.
- Jobarteh, M.L., Moore, S.E., Kennedy, C., Gambling, L., and McArdle, H.J. (2014) The effect of delay in collection and processing on RNA integrity in human placenta: experiences from rural Africa. Placenta. 35:1, 72–74.