Variation of Peripheral Blood Mononuclear Cell RNA Quality in Archived Samples

Abstract

The Infectious Diseases BioBank (IDB) has consistently archived peripheral blood mononuclear cell (PBMNC) RNA for transcriptome analyses. RNA is particularly labile, and hence, these samples provide a sensitive indicator for assessing the IDB's quality-assurance measures. Independent analyses of 104 PBMNC RNA specimens from 26 volunteers revealed that the mean RNA integrity number (RIN) was high (9.02), although RIN ranged between scores of 7 and 10. This variation of RIN values was not associated with ischemic time, PBMNC quality, number of samples processed per day, self-medication after immunization, freezer location, donor characteristics, differential white blood cell counts, or daily variation in RNA extractions (all P>0.05). RIN values were related to the date of collection, with those processed during mid-summer having highest RIN scores (P=0.0001). Amongst specimens with the lowest RIN scores, no common feature could be identified. Thus, no technical explanation for the variation in RNA quality could be ascertained and these may represent normal physiological variations. These data provide strong evidence that current IDB protocols for the isolation and preservation PBMNC RNA are robust.

FIG. 1.

Examples of RNA quality data. Left: Representative electrophoresis of peripheral blood mononuclear cell RNA. Right: Corresponding fluorescent traces used to derive RIN values (of 7.5, 9.2, 9.5, 8.9, 9.3, and 9.4, respectively, for human immune response dynamics samples 001-1, 001-2, 001-3, 001-4, 005-1, and 005-2 illustrated left to right on the gel photograph). RIN, RNA integrity number.

FIG. 2.

RNA quality versus ischemic time.

FIG. 3.

RNA quality as a function of collection date in 2010. RIN values were plotted against the date of processing/freezing, with data analyzed to determine the line of best fit.