RNA integrity and the effect on the real-time qRT-PCR performance.
The assessment of RNA integrity is a critical first step in obtaining meaningful gene expression data. Working with low-quality RNA may strongly compromise the experimental results of downstream applications which are often labour-intensive, time-consuming, and highly expensive. Using intact RNA is a key element for the successful application of modern molecular biological methods, like qRT-PCR or micro-array analysis. To verify RNA quality nowadays commercially available automated capillary-electrophoresis systems are available which are on the way to become the standard in RNA quality assessment. Profiles generated yield information on RNA concentration, allow a visual inspection of RNA integrity, and generate approximated ratios between the mass of ribosomal sub-units. In this review, the importance of RNA quality for the qRT-PCR was analyzed by determining the RNA quality of different bovine tissues and cell culture. Independent analysis systems are described and compared (OD measurement, NanoDrop, Bioanalyzer 2100 and Experion). Advantage and disadvantages of RNA quantity and quality assessment are shown in performed applications of various tissues and cell cultures. Further the comparison and correlation between the total RNA integrity on PCR performance as well as on PCR efficiency is described. On the basis of the derived results we can argue that qRT-PCR performance is affected by the RNA integrity and PCR efficiency in general is not affected by the RNA integrity. We can recommend a RIN higher than five as good total RNA quality and higher than eight as perfect total RNA for downstream application.
Fleige
Simone
S
Physiology Weihenstephan, Center of Life and Food Sciences (ZIEL), Technical University of Munich, 85350 Freising, Germany.
Pfaffl
Michael W
MW
D000818:Animals; D003199:Computer Systems; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D012313:RNA / Q000737:chemistry* / Q000302:isolation & purification / Q000592:standards; D012333:RNA, Messenger / Q000737:chemistry* / Q000592:standards; D012680:Sensitivity and Specificity; D012984:Software
D012333:RNA, Messenger; D012313:RNA