RNA Quality Assessment
a. Quantitative analysis
GenCore uses Thermo Nanodrop2000, Invitrogen Qubit and Molecular device SpectraMax PicoGreen instruments for quantitation of nucleic acids. Nanodrop spectrophotometer is used for UV spectrophotometric measurements to determine the RNA concentration and optical density (OD) 260/280 ratio. The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of RNA. A ratio of >1.8 and <2.2 is generally accepted as pure RNA, deviations from these range is usually indicative of contaminants. However, the quality of RNA cannot be definitely judged by Nanodrop and a gel-based approach is needed to assess the quality of RNA.
The RNA concentration is determined by measuring the absorbance at 260 nm on the Nanodrop using 1 ul of sample. The Nanodrop reports concentration of individual samples as ng/μl, which is calculated using the relationship; absorbance of 1 unit at 260 nm corresponds to 40 ug of RNA per ml. After the concentration determination, the RNA quality is assessed using a Bioanalyzer on a RNA specific chip. The Yerkes GenCore also uses Qubit and PicoGreen methods for RNA quantitation. Qubit and PicoGreen offers high specificity and selectivity for RNA molecules over the Nanodrop. This feature allows for accurate quantitation of samples. Qubit offers flexibility when DNA or phenol contamination is suspected in the sample.
b. Qualitative analysis
The Agilent 2100 Bioanalyzer is a microfluidics based platform used for sizing and quality assessment of Nucleic acids at GenCore. The Bioanalyzer is the industry standard for assessing the quality of RNA based on its patented RNA integrity number (RIN) system. The RIN is a software algorithm designed to estimate the integrity of the RNA; the software automatically generates a numerical value (RIN score) for each RNA sample based on its entire electrophoretic trace. The software assigns a numerical value to each sample ranging from 1 to 10, with 1 being totally degraded RNA and 10 being highly intact RNA. Figure 1 illustrates poor quality RNA sample with RIN score 2.6 (Fig. 1A), good quality RNA with RIN score 10 (Fig. 1B), cDNA amplified from single cell using the Clontech SmartER kit (Fig. 1C), where size range of full length cDNAis 500-over 10,000 nt and library constructed using Illumina TruSeq RNA kit, where the average library size is approximately 300 nt. (panel D).
Figure 1: A. Bioanalyzer electrophoretic traces for poor quality RNA with RIN score 2.60, B. Good quality RNA with RIN score 10, C. cDNA amplified using Clontech SmartER technology, D. TruSeq Stranded library.
RNA integrity will be determined using the Agilent 2100 Bioanalyzer on a RNA 6000 Nano LabChip® or RNA 6000 Pico LabChip®. Next generation sequencing protocols consistently require the RNA to be of very high quality with RIN scores over 8, especially for mRNA based library prep methods.
Following the initial quantitative and qualitative analysis of the sample, if there is any concern about the sample(s) the investigator will be contacted promptly to decide whether to proceed, replace or drop such sample(s).