Medeiros M, Sharma V K, Ding R, Yamaji K, Li B, Muthukumar T, Valderde-Rosas S, Hernandez A M, Muñoz R, Suthanthiran M
Weill Medical College of Cornell University, New York, NY, USA.
J Immunol Methods. 2003 Aug;279(1-2):135-42. doi: 10.1016/s0022-1759(03)00237-0.
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.
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.
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.
The addition of RNAlater to urine cell pellets improves RNA isolation from urinary cells and facilitates measurement of low abundance mRNAs.
我们已经表明，测量尿细胞中细胞毒性攻击蛋白穿孔素和颗粒酶B的mRNA是诊断人类肾移植急性排斥反应的一种非侵入性方法。尿细胞mRNA研究在其他患者群体（如癌症患者）中也产生了有用的信息。然而，从尿细胞中分离出足够数量和高质量的核糖核酸（RNA）存在问题。据报道，RNA稳定剂RNAlater可优化从室温保存的肿瘤组织和富含色素的眼组织中分离RNA。
我们探讨了向尿细胞沉淀中添加RNAlater是否能提高RNA产量、增强纯度并便于测量低丰度mRNA。我们使用实时定量聚合酶链反应（PCR）测定法，测量了从肾移植受者获得的尿液标本和肾活检组织中组成性表达基因18S rRNA以及颗粒酶B和转化生长因子-β(1)（TGF-β(1)）mRNA的表达水平。
与速冻前未用RNAlater处理的细胞沉淀相比，速冻前用RNAlater处理的尿细胞沉淀的RNA产量（P<0.01，Wilcoxon符号秩检验）和A260/A280比值（P<0.01）均更高。与未处理的细胞沉淀相比，处理后的尿细胞沉淀中18S rRNA（P<0.02）、颗粒酶B mRNA（P=0.002）和TGF-β(1)（P=0.02）的水平（每1微克总RNA的拷贝数）均更高。Kruskall-Wallis单向方差分析以及与Student-Newman-Keuls检验的成对比较表明，肾移植活检组织与未处理的尿细胞沉淀之间颗粒酶B（P<0.05）和TGF-β(1)（P<0.05）的mRNA水平存在显著差异，但活检标本与用RNAlater处理的尿细胞沉淀之间无显著差异。
向尿细胞沉淀中添加RNAlater可改善从尿细胞中分离RNA的效果，并便于测量低丰度mRNA。
