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

Author(s): Bradley MS, Boudreau MH, Grenier C, Huang Z, Murphy SK, Siddiqui NY

Publication: Female Pelvic Med Reconstr Surg, 2017, Vol. , Page

PubMed ID: 29194081 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of delayed RNA extraction and type of extraction method used on RNA yield and integrity. Fresh-voided midstream clean-catch urine was collected from 10 female patients and stored at 4˚C. Four aliquots of the specimen were centrifuged at 1600 x g for 10 min and resuspended in RNA later, while the remaining specimen was pushed through a ZRC GF filter and combined with ZR Urine RNA isolation buffer. Specimens were transported to the laboratory on cold packs in RNAlater or RNA isolation buffer. Upon arrival, RNA was isolated from specimens in RNAlater using TRI Reagent or the Absolutely RNA Nanoprep Kit and from the specimen in RNA isolation buffer using the ZR Urine RNA Isolation Kit with combined DNAse treatment. RNA was quantified using the Nanodrop spectrophotometer and by real-time PCR amplification of B2M and GAPDH using TaqMan probes. The effect of delayed processing on RIN was further studied using nine additional specimens extracted using the ZR Urine RNA isolation kit 146-235 min after collection.

   Summary of Findings:

   The yield was significantly higher when RNA was extracted using TRI Reagent than with the Absolutely RNA Nanoprep (P=0.028) or ZR Urine RNA isolation kit (P=0.009), but yields were comparable between the Absolutely RNA Nanoprep and ZR Urine RNA isolation Kit. However, the A260/280 ratios were significantly lower for RNA extracted with TRI Reagent than with the Absolutely RNA Nanoprep kit (P=0.047). GAPDH CT values were significantly lower when RNA was extracted with the ZR Urine RNA isolation kit rather than with the Absolutely RNA Nanoprep kit (P = 0.012) or TRI Reagent (P=0.028). The CT values for B2M were lowest when RNA was extracted using the ZR Urine RNA isolation kit, but the difference was not significant. The time between collection and RNA isolation had no effect on RNA yield or RIN.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	75-195 min 146-235 min
   Analyte Extraction and Purification	Analyte isolation method	TRI Reagent Absolutely RNA Nanoprep kit ZR Urine RNA isolation kit

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