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Urine RNA Processing in a Clinical Setting

Comparison of 3 Protocols

Bradley, Megan S., MD*; Boudreau, Marie-Helene; Grenier, Carole, BS; Huang, Zhiqing, PhD; Murphy, Susan K., PhD; Siddiqui, Nazema Y., MD, MSc

Female Pelvic Medicine & Reconstructive Surgery: May/June 2019 - Volume 25 - Issue 3 - p 247–251
doi: 10.1097/SPV.0000000000000525
Original Articles
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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.

The ZR Urine RNA Isolation Kit provided the highest quality of extracted RNA from urine samples collected in a clinical setting.

From the *Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Hospital, University of Pittsburgh School of Medicine, Pittsburgh, PA;

Department of Obstetrics and Gynecology, and

Division of Urogynecology and Reconstructive Pelvic Surgery, Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC.

Correspondence: Megan S. Bradley, MD, Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Hospital, University of Pittsburgh School of Medicine, 300 Halket St, Pittsburgh, PA 15217. E-mail: Megsbrad2010@gmail.com.

Dr. Siddiqui has protected research time and provided support according to her award number K12-DK100024 from the National Institute of Diabetes and Digestive and Kidney Diseases. Dr. Siddiqui has received grant funding from Medtronic Inc, but this resource did not provide support for this study. Additional funding was obtained from the Duke University Hammond Research Fund. The other authors did not report any potential conflicts of interest.

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