RNA quality assessment is a foundational step in molecular biology experiments; ensuring integrity is necessary because degraded samples can lead to inconsistent or failed experimental results, wasting time and costly reagents. Traditional metrics work well for pristine samples, but modern research frequently involves challenging inputs, necessitating specialized quality control methods. The DV200 metric has emerged as a specialized tool for assessing the fragmentation of these difficult RNA samples.
Defining the DV200 Metric
The DV200 metric is a precise measure of RNA fragmentation, defined as the percentage of total RNA fragments greater than 200 nucleotides (nt) in length. This index provides a direct quantitative assessment of usable material, especially when the RNA is already degraded. Fragments shorter than the 200-nucleotide cutoff are generally poor substrates for molecular applications, such as sequencing library preparation, and may be lost during purification.
This metric is generated using automated capillary electrophoresis systems, such as the Agilent Bioanalyzer or TapeStation. The system runs the RNA sample through microfluidic channels, separating the molecules by size and producing an electropherogram. Specialized software performs a “smear analysis” on this trace, integrating the area under the curve for all fragments between 200 nt and 10,000 nt. The final DV200 value is calculated by dividing this integrated area by the total area under the entire RNA trace, expressing the result as a percentage.
DV200 Versus RNA Integrity Number (RIN)
The standard metric for RNA quality has long been the RNA Integrity Number (RIN), which ranges from 1 (most degraded) to 10 (most intact). The RIN score is calculated by analyzing the ratio of the 28S and 18S ribosomal RNA (rRNA) peaks in the electropherogram. For high-quality, intact RNA, the 28S peak is approximately twice the height of the 18S peak, resulting in a high RIN score.
The RIN system fails to accurately assess quality for samples that are inherently fragmented or heavily processed, such as those derived from Formalin-Fixed Paraffin-Embedded (FFPE) tissues. Formalin fixation causes chemical cross-linking and degradation, breaking down rRNA molecules and eliminating the distinct 28S and 18S peaks the RIN algorithm relies on. Highly degraded FFPE samples often yield a low RIN score (typically 2 to 4), which lacks the resolution to differentiate between usable and unusable material. The DV200 was developed to overcome this limitation, offering a reliable quality measure for fragmented samples where RIN is ineffective.
Practical Applications and Required Thresholds
The DV200 metric is the preferred quality control standard for Next-Generation Sequencing (NGS) when working with challenging inputs like FFPE tissues, liquid biopsies, or old clinical archives. It is a stronger predictor of successful sequencing library preparation than the RIN score in these contexts. A higher DV200 score correlates directly with a higher yield of the final sequencing library and a greater percentage of reads that can be successfully mapped to the genome.
Specific molecular applications require different DV200 thresholds, which are used to fine-tune the amount of input RNA required for library preparation. For standard RNA sequencing of highly fragmented FFPE samples, a minimum DV200 value of 30% is generally considered the lowest acceptable threshold. For specialized, high-sensitivity protocols, such as single-cell analysis, a DV200 score of 30% is a common cut-off point. A DV200 value above 66% has been shown to be an optimal cutoff for predicting a sufficient amount of first-strand complementary DNA product.
Maximizing RNA Quality Through Sample Handling
Achieving a high DV200 score depends on careful pre-analytic sample handling to minimize RNA degradation by endogenous enzymes. A primary factor is cold ischemia time, the duration between tissue excision and the start of fixation or freezing. Optimal protocols require fixation or stabilization to begin within 30 minutes of tissue removal to prevent nucleases from breaking down the RNA.
Tissue must be stored properly, typically at -80°C for long-term preservation, or stabilized immediately after collection using commercial solutions. The extraction process requires specialized kits designed to reverse formalin-induced cross-linking and minimize further fragmentation. Adhering to strict RNase-free working standards, including the use of decontaminated surfaces and dedicated reagents, is necessary to prevent environmental contamination that could lower the final DV200 score.