Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Urinary cell mRNA profiling of kidney allograft recipients: A systematic investigation of a filtration based protocol for the simplification of urine processing.

Author(s): Snopkowski C, Salinas T, Li C, Stryjniak G, Ding R, Sharma V, Suthanthiran M

Publication: J Immunol Methods, 2021, Vol. 498, Page 113132

PubMed ID: 34464607 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared RNA yield, purity, and the number of amplifiable copies (i) between RNA isolated from the urine of kidney allograft recipients using a filtration-based method (Zymo filter-based protocol, ZFBP) and the Cornell centrifugation-based protocol (CCBP), (ii) between RNA from Zymo filtrates that were stored at room temperature compared versus those stored at  -80°C for 2-4 days, (iii)  filtrates shipped at ambient temperature versus those stored at -80°C, and (iv) urine specimens that were processed at home versus in the laboratory. The authors also compared levels of constitutively expressed RNA and rejection markers (granzyme B and perforin) in the urine of allograft recipients with biopsy-confirmed acute rejection and those without rejection. Urine was collected from kidney allograft recipients. To compare the isolation methods, urine (47 specimens from 39 patients) was divided into two aliquots from which the RNA was extracted using the CCBP and ZFBP methods within 4-6 h of urine collection.  The CCBP protocol pelleted urinary cells by centrifugation at 2000 g for 30 min, resuspended the sediment in Buffer RLT, stored the lysate -80°C, and extracted the RNA using the RNeasy mini Kit. For the ZFBP method, the urine was pushed with a syringe through a Zymo filter, the filtrate was resuspended in Urine Extraction Buffer, and stored at -80°C until RNA extraction using the Zymo Urine RNA Isolation Kit. Unless otherwise specified, urine was pushed with a syringe through a Zymo filter, the filtrate was resuspended in Urine RNA Buffer, and the filtrate was stored at -80°C until RNA extraction using the Zymo Urine Isolation Kit Version 2.0. To investigate the potential effects of filtrate storage, the resuspended filtrate from the urine from 12 kidney allograft recipients was stored at either -80°C or room temperature for 2-4 days before RNA extraction. To investigate the effect of shipping filtrate, resuspended filtrate from pooled urine specimens from 12 kidney allograft recipients (three pools from 3-5 specimens) was stored at -80°C or shipped at ambient temperature before RNA extraction. To investigate the effects of patients processing the filtrate at home versus processing in the lab, five kidney allograft recipients self-collected, filtered, resuspended, and stored twenty-five urine filtrates at room temperature overnight the night before clinic visits. The same allograft recipients provided a fresh specimen the next day at the clinic, which was processed by the clinic staff. Both filtrates were then frozen at -80°C until RNA extraction. To investigate if urine can be used to identify acute allograft rejection, urine specimens matched to eight biopsies with acute rejection and fourteen biopsies without rejection (representing a total of 20 allograft recipients) were compared. Allograft rejection was diagnosed by a pathologist based on percutaneous core needle biopsy and characterized as acute cellular or acute cellular with antibody mediated rejection. RNA was quantified using a NanoDrop spectrophotometer and, in select specimens, using a Qubit fluorometer. 18S rRNA (reference), TGF-β1 (constitutive expressed), perforin (rejection marker), granzyme B (rejection marker), and/or miR-26 were quantified by real-time RT-PCR. Rejection was diagnosed by a pathologist based on percutaneous core needle biopsy of the allograft. 

   Summary of Findings:

   Significantly more RNA was obtained using the Zymo-based method than the CCBP method when all specimens were considered (0.81 versus 0.30 µg, p<0.0001) and when only the 38 specimens with equal volumes were considered (0.47 versus 0.15 µg, p<0.0001). The purity of RNA (A260/A280) was comparable between the extraction methods evaluated. All specimens yielded adequate amplifiable rRNA (>5 x 107 copies 18S rRNA per µg RNA), and the number of amplifiable copies of 18S rRNA did not differ between extraction methods. When the filtrate was stored at room temperature for 2-4 days rather than -80°C, the median RNA yield was slightly higher by spectrophotometer (0.48 versus 0.40 µg, p=0.04) but there were no differences in RNA yield by fluorometer (0.20 versus 0.23 µg, p=0.30), spectrophotometric purity (A260/A280 of 1.80 versus 1.87, p=0.57), or the number of amplifiable copies per microgram RNA of 18S rRNA (1.13 x 109 versus 8.18 x 108, p=0.24), TGF-β1 (3.44 x 103 versus 4.76 x 103, p=0.08), or miR-26a (8.34 x 105 versus 9.17 x 108, p=0.58). RNA extracted from filtrates mailed at ambient temperature and those stored -80°C had comparable median RNA yield by spectrophotometer (1.59 versus 1.83 µg, p=0.25) or fluorometer (1.01 versus 1.07 µg, p>0.99), spectrophotometric purity (A260/A280 of 2.01 versus 2.04, p=0.50), and amplifiable copies per microgram RNA of 18S rRNA (2.70 x 108 versus 2.36 x 108, p>0.99), TGF-β1 (2.87 x 103 versus 1.68 x 103, p=0.75) and miR-26a (2.36 x 106 versus 2.82 x 106, p=0.75). RNA extracted from filtrates processed by the patient at home and those processed by laboratory staff had comparable median RNA yield by spectrophotometer (1.66 versus 2.12 µg, p=0.75) or fluorometer (0.89 versus 1.57 µg, p=0.47), spectrophotometric purity (A260/A280 of 1.97 versus 1.99, p=0.57), and amplifiable copies per microgram RNA of 18S rRNA (3.97 x 108 versus 3.24 x 108, p=0.44) and TGF-β1 (7.03 x 102 versus 3.46 x 102, p=0.37).  When specimens from allograft recipients with acute rejection and those without were compared, five specimens were excluded: two due to insufficient urine volume (one from a recipient with acute rejection and one from a recipient without rejection) and three due to insufficient TGF-β1 copy number (one from recipient with acute rejection and two from recipients without rejection). In the remaining specimens (6 specimens from patients with acute rejection and 11 specimens from patients without rejection), there were comparable copies of amplifiable18S rRNA (2.3 x 109 versus 1.93 x 109, p=1.00) and TGF-β1 (2760 versus 4150, p=0.81), but RNA extracted from the urine of recipients with acute rejection had more copies per microgram RNA of the rejection markers granzyme B (7415 versus 694, P=0.01) and perforin (1029 versus 111, p=0.00002).

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Fluorometry
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	ZFBP method (pushed through a Zymo filter, resuspension in Urine Extraction Buffer, storage at -80°C, and extraction using the Zymo Urine RNA Isolation Kit) CCBP (centrifugation at 2000 g for 30 min, resuspension in Buffer RLT, storage at -80°C and extraction using the RNeasy mini kit)
   Preaquisition	Diagnosis/ patient condition	Kidney allograft with acute rejection Kidney allograft without acute rejection
   Biospecimen Acquisition	Locale of biospecimen collection	Collection and processing to filtrate at home Collection and processing to filtrate at the clinic
   Storage	Storage temperature	Room temperature -80°C
   Storage	Between site transportation method	Mailed Not transported

   View more