DV200 Index for Assessing RNA Integrity in Next-Generation Sequencing

Abstract

Poor quality of biological samples will result in an inaccurate analysis of next-generation sequencing (NGS). Therefore, methods to accurately evaluate sample integrity are needed. Among methods for evaluating RNA quality, the RNA integrity number equivalent (RINe) is widely used, whereas the DV200, which evaluates the percentage of fragments of >200 nucleotides, is also used as a quality assessment standard. In this study, we compared the RINe and DV200 RNA quality indexes to determine the most suitable RNA index for the NGS analysis. Seventy-one RNA samples were extracted from formalin-fixed paraffin-embedded tissue samples (n = 30), fresh-frozen samples (n = 25), or cell lines (n = 16). After assessing RNA quality using the RINe and DV200, we prepared two kinds of stranded mRNA sequencing libraries. Finally, we calculated the correlation between each RNA quality index and the amount of library product (1 st PCR product per input RNA). The DV200 measure showed stronger correlation with the amount of library product than the RINe (R 2 = 0:8208 for the DV200 versus 0.6927 for the RINe). Receiver operating characteristic curve analyses revealed that the DV200 was the better marker for predicting efficient library production than the RINe using a threshold of >10 ng/ng for the amount of the 1 st PCR product per input RNA (cutoff value for the RINe and DV200, 2.3 and 66.1%; area under the curve, 0.99 and 0.91; sensitivity, 82% and 92%; and specificity, 93% and 100%, respectively). Our results indicate that NGS libraries prepared using RNA samples with the DV200 value > 66:1% exhibit greater sensitivity and specificity than those prepared with the RINe values > 2:3. These findings suggest that the DV200 is superior to the RINe, especially for low-quality RNA, because it is a more consistent assessment of the amount of the 1 st NGS library product per input.

Genetic Signatures From RNA Sequencing of Pediatric Localized Scleroderma Skin

“…RNAs were quantified using a Nanodrop ND-100 Spectrophotometer (Nanodrop Technologies, Wilmington, USA) and a 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit, Waldbronn, Germany). Extracted RNA samples were only sequenced if they had a %DV200 (percentage of RNA fragments > 200 nucleotides) ( 27 ) >30% for FFPE samples and an RNA integrity number of ≥8 for RNAlater samples to ensure quality control.…”

Analytic validation and clinical utilization of the comprehensive genomic profiling test, GEM ExTra®

“…Tumor types included astrocytoma, colon, GBM, GIST, lung, lymphoma, melanoma, neuroblastoma, ovarian, pancreas, sarcoma, stomach and urothelial. Minimum inputs were used for each replicate (50 ng DNA and RNA input based on DV200 score [ 22 ]). Correlation acceptability was set at an average >90% agreement of all (repeatability and reproducibility) replicates.…”

Protocol Improvement for RNA Extraction From Compromised Frozen Specimens Generated in Austere Conditions: A Path Forward to Transcriptomics-Pathology Systems Integration

“…The RIN values were consistently around 4 and DV 200 values were lower than 30; and these suboptimal scores could be attributed to the compound effects of several factors, including the compromised tissue preservation method and the effect on RNA quality due to NBF (cross-linking and direct reaction with nucleotides; Cox et al, 2006 ). By certain account, the DV 200 score is the fittest metric to decide upon the appropriateness of the samples to run for gene expression analysis (Matsubara et al, 2020 ); while the prevalent thought is to consider both RIN and DV 200 to finalize this decision. Using RNA samples with RIN score >2.0, Ravo et al claimed a complete success in microarray-based cDNA-mediated annealing, selection, extension and ligation (DASL) assay; furthermore, these authors achieved similar success in DASL assays using samples with RIN <2.0 but the majority of RNA size was >200 nt (Ravo et al, 2008 ).…”