Urine RNA Processing in a Clinical Setting: Comparison of 3 Protocols

Abstract

Objective: The objective of this study was to compare quantitative and qualitative RNA extraction results from clinical voided urine samples between 3 commercially available extraction protocols.

Methods: For phase 1, fresh voided urine samples from 10 female subjects were collected and processed in clinic and transported to the laboratory with cold packs. RNA was purified with 1 of 3 RNA extraction protocols: (1) TRI Reagent Protocol; (2) Absolutely RNA Nanoprep Kit; and (3) ZR Urine RNA Isolation Kit. Real-time polymerase chain reactions (RT-PCR) were performed. As the ZR Urine RNA Isolation Kit provided the highest quality RNA in phase 1, for phase 2, RNA was extracted from 9 additional voided urine specimens using this kit to perform additional qualitative analyses.

Results: Median RNA yield was significantly higher with the TRI Reagent Protocol as compared with the other protocols (P = 0.007). However, there was a significantly lower median threshold cycle value from polymerase chain reaction (indicating improved downstream application performance) with the ZR Urine RNA Isolation Kit as compared with the other methods (P = 0.005). In phase 2, the median RNA integrity number of urine RNA was 2.5 (range, 1.6-5.9).

Conclusions: Although other methods may provide a higher quantity of RNA, when using clinical urine samples, the ZR Urine RNA Isolation Kit provided the highest quality of extracted RNA. This kit is especially attractive for the clinical setting because it does not require an initial centrifugation step. The urine RNA obtained with this kit may be useful for polymerase chain reaction but is not likely to be of high enough integrity for RNA sequencing.

Figure 1

Study Flow Diagram

Figure 2

mRNA evaluation by specific RT-PCR with GAPDH and B2M by extraction method. Median Threshold Cycle (C_t) value presented from duplicate (n=10) GAPDH, Glyceraldehyde-3-phosphate dehydrogenase, B2M, β2 microglobulin *Median C_t values compared with Friedman test, post-hoc analysis with Wilcoxan Signed-Rank test

Figure 3

Agilent 4200 TapeStation capillary electrophoretic profiles of two RNA samples processed with ZR Urine Isolation Kit: (A) RNA integrity score 3.3 and (B) RNA integrity score 5.9 * The RNA Integrity (RIN) algorithm is applied to electrophoretic RNA measurements, typically obtained using capillary gel electrophoresis, and based on a combination of different features. When interpreting RIN scores, a score of 1 suggests the most degraded profile and 10 being the most intact.

Figure 3

Agilent 4200 TapeStation capillary electrophoretic profiles of two RNA samples processed with ZR Urine Isolation Kit: (A) RNA integrity score 3.3 and (B) RNA integrity score 5.9 * The RNA Integrity (RIN) algorithm is applied to electrophoretic RNA measurements, typically obtained using capillary gel electrophoresis, and based on a combination of different features. When interpreting RIN scores, a score of 1 suggests the most degraded profile and 10 being the most intact.