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Extracting RNA from urine cells

Extracting RNA from urine cells - RNA Techniques Forum

Extracting RNA from urine cells - Post and discuss RNA methods and RNA science related topics. RNA extraction, cDNA synthesis, RNA EMSA, and other RNA protocols.


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Old 02-19-2006, 12:55 PM
Pipette Filler
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Default Extracting RNA from urine cells



Dear Friends,
I'm trying to isolate good quality RNA from urine cells. I'm facing troubles mainly in the cell isolation protocol (e.g. 500 g 5 min 4 deg vs. 15,000g 20 min RT). The RNA is poor and degraded. Does anyone has a good (and prooved) protocol for cell isolation and RNA extraction from urine?

Thanks

Gal
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Old 02-20-2006, 07:27 AM
Pipette Filler
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I was stumped by the urine cell analysis however I found this from pubmed search on molecularstation.com

from paper:

Cancer Lett. 2006 Feb 10; [Epub ahead of print] Related Articles, Links
Click here to read Click here to read
Urinary cytokeratin 20 mRNA expression has the potential to predict recurrence in superficial transitional cell carcinoma of the bladder.

Christoph F, Weikert S, Wolff I, Schostak M, Tabiti K, Muller M, Miller K, Schrader M.

Department of Urology, Universitatsmedizin Berlin, Charite-Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany.

Higher levels of cytokeratin 20 (CK 20) mRNA are expressed in malignant urothelial tissue compared to normal tissue. We determined the CK 20 mRNA expression in urine from patients with transitional cell carcinoma (TCC) of the bladder and assessed the biological behavior of such tumors in a 5-year follow-up. Second voided urine was preoperatively collected from 56 patients with bladder carcinoma, from 20 patients with nonmalignant urological diseases and from 40 healthy volunteers. RNA extraction from exfoliated urothelial cells was followed by quantitative real-time RT-PCR with the Light Cycler((R)). Patients in the superficial TCC group had a median expression of 8226AU (arbitrary units) with and 1523AU without tumor recurrence (P=0.023). No such correlation was detected in the group with muscle-invasive tumors. Kaplan-Meier analysis revealed a significant difference between recurrent and nonrecurrent disease (P=0.019) in superficial but not in muscle-invasive TCC (P=0.84). CK 20 mRNA expression in urine has the potential to identify patients at risk for recurrence of noninvasive papillary urothelial tumors. It helps to categorize patients prior to TUR-B, so that the cystoscopy interval during follow-up may be extended in those with low-risk superficial TCC.

2. Materials and methods

This study included 56 patients (19 women and 37 men) with superficial and muscle-invasive disease treated at the Department of Urology between October 1997 and January 2000. All patients with superficial disease underwent transurethral resection of the bladder (TUR-B), whereas those with muscle-invasive disease were submitted to radical cystectomy. Histopathological staging and grading were done according to the TNM classification of the International Union Against Cancer [18]. All patients signed a consent form approved by the Committee on Human Rights in Research of our institution. Urine control samples were taken from 20 patients with nonmalignant urological diseases (benign prostatic hyperplasia, urolithiasis, cystitis) as well as from 40 healthy volunteers.
2.1. Sample preparation

Second voided urine samples of 100 ml were obtained from each patient prior to the initial TUR-B. The urine was centrifuged, and the cells were washed with phosphate-buffered saline (PBS–Dulbecco, without calcium/magnesium).
2.2. RNA extraction

Total RNA was extracted from urine samples using a commercially available RNA extraction kit (Trizol, Gibco, Carlsbad, California) according to the manufacturer's instructions. To avoid contamination by DNA during RNA preparation, RNA was digested with RNAse-free DNAse I (Gibco), as recommended by the supplier.

2.3. RT-PCR

Detection of CK 20 mRNA was conducted by a two-step procedure using the Light Cycler CK 20 mRNA Quantification Kit (Roche, Germany). First, cDNA was synthesized with a reverse transcription kit (Reverse Transcriptase Master Mix, Roche Diagnostics, Germany) using 5 μl of RNA and random hexamers serving as primers in a total volume of 20 μl.

What type of cells are you trying to examine? epithelial cells or white/red blood cells in the case of hematuria?
Maybe you can FAC sort them to get more specific cell types?
Cheers, hope that helps,
Dave

Last edited by dave2006; 02-20-2006 at 07:32 AM.
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