Optimization of RNA yield, purity and mRNA copy number by treatment of urine cell pellets with RNAlater.
BACKGROUND
We have shown that measurement of mRNA for cytotoxic attack proteins perforin and granzyme B in urinary cells is a noninvasive means of diagnosing acute rejection of human renal allografts. Urinary cell mRNA studies have yielded useful information in other patient populations such as patients with cancer. The isolation of sufficient and high quality ribonucleic acid (RNA) from urinary cells however is problematic. RNAlater, an RNA stabilization solution, has been reported to optimize RNA isolation from tumor tissues stored at room temperature and from pigment-rich ocular tissues.


METHODS
We explored whether the addition of RNAlater to urine cell pellets improves RNA yield, enhances purity and facilitates measurement of low abundance mRNAs. We measured, with the use of real-time quantitative polymerase chain reaction (PCR) assay, levels of expression of a constitutively expressed gene 18S rRNA and mRNA for granzyme B and transforming growth factor-beta(1) (TGF-beta(1)) in urine specimens and renal biopsies obtained from renal allograft recipients.


RESULTS
RNA yield (P<0.01, Wilcoxon signed rank test) and the A260/A280 ratio (P<0.01) were both higher with urine cell pellets treated with RNAlater prior to snap freezing compared to cell pellets that were not treated with RNAlater prior to snap freezing. Levels (copy number per 1 microg of total RNA) of 18S rRNA (P<0.02), granzyme B mRNA (P=0.002) and TGF-beta(1) (P=0.02) were all higher with treated urine cell pellets compared to untreated cell pellets. Kruskall-Wallis one way analysis of variance and pair-wise comparisons with Student-Newman-Keuls test showed that the levels of mRNA for granzyme B (P<0.05) and TGF-beta(1) (P<0.05) are significantly different between renal allograft biopsies and untreated urine cell pellets but not between the biopsy specimens and RNAlater-treated urine cell pellets.


CONCLUSIONS
The addition of RNAlater to urine cell pellets improves RNA isolation from urinary cells and facilitates measurement of low abundance mRNAs.
Medeiros
M
M
Weill Medical College of Cornell University, New York, NY, USA.
Sharma
V K
VK
Ding
R
R
Yamaji
K
K
Li
B
B
Muthukumar
T
T
Valderde-Rosas
S
S
Hernandez
A M
AM
Muñoz
R
R
Suthanthiran
M
M
D000293:Adolescent; D002648:Child; D053804:Granzymes; D006801:Humans; D012313:RNA / Q000032:analysis / Q000302:isolation & purification* / Q000652:urine; D012337:RNA, Ribosomal, 18S / Q000652:urine; D012697:Serine Endopeptidases / Q000235:genetics; D013053:Spectrophotometry; D016212:Transforming Growth Factor beta / Q000235:genetics; D014556:Urine / Q000166:cytology*
D012337:RNA, Ribosomal, 18S; D016212:Transforming Growth Factor beta; D012313:RNA; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases