Name: Urine Exfoliated Cell and Bacteria RNA Purification Kit
Brand: Norgen Biotek Corp.
SKU: 22550
Price: 239.000000 USD
Availability: InStock

Features and Benefits

Purify all sizes of RNA (including microRNA) without the need for phenol
Isolate and detect total RNA from 1 mL and up to 50 mL urine
Provides high-quality RNA for sensitive applications - isolate RNA from as little as 100 cells
Rapid processing time from sampling to downstream testing
Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

This kit provides a rapid spin column method for the isolation and purification of total RNA (including microRNA) from exfoliated cells that have been shed into the urine from the urinary tract. The kit purifies all sizes of RNA, from large mRNA and ribosomal RNA down to microRNA (miRNA) and small interfering RNA (siRNA). The RNA is preferentially purified from other cellular components such as proteins, as well as from the contaminating species found in urine such as glucose and salts, without the use of phenol or chloroform. The purified RNA is of the highest integrity and can be used in a number of downstream applications including real-time RT-PCR, qRT-PCR, Northern blotting, NGS, and expression array assays. Multiple samples can be processed in 20 minutes.

Background

RNA biomarkers from exfoliated cells can be used as non-invasive tools for a number of diagnostic and research applications including the diagnosis and monitoring of bladder, kidney, or other urinary-tract cancers.

Details

Supporting Data

Figure 1. Isolation and Detection of RNA from the Exfoliated Cells in 50 mL of Urine. RNA was isolated from the exfoliated cells in a 50 mL urine sample obtained from a healthy male without any spiking using Norgen's Urine (Exfoliated Cell) RNA Purification Kit. The bind, wash, and elute procedure was followed, and the purified RNA was recovered in 20 µL of the Elution Buffer. A dilution series of HeLa cells with known concentration (from 1 cell per mL to 1,000 cells per mL) was isolated in parallel as standards. Seven microliters of the 20 µL eluted RNA were then subjected to a 20 µL reverse transcription. One microliter of the reverse transcription was used in a 20 µL real-time PCR reaction with primers to detect the ribosomal S14 transcript. The red lines in the PCR baseline graph above correspond to the HeLa RNA standards, while the blue lines correspond to the successful PCR results when RNA isolated from the exfoliated cells in 50 mL of non-spiked urine was used as the template.

Kit Specifications
Maximum Column Binding Capacity	50 μg
Volume of Urine Processed	1-50 mL
Maximum Input of Exfoliated Cells	1 x 10⁶
Size of RNA Purified	All sizes, including small RNA (< 200 nt)
Time to Complete 10 Purifications	20 minutes (for Exfoliated Cells) 30 minutes (for Bacteria)
Average Yield	~1μg RNA per 1 x 10⁵ cells (For Exfoliated Cells - Varies due to cell density sample) ~0.5μg RNA per 1 x 10⁷ cells (For Bacteria - Varies due to cell density sample)

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.

Component	Cat. 22550 (25 preps)
Buffer SK	15 mL
Wash Solution A	18 mL
Elution Buffer E	6 mL
Micro Spin Columns	25
Collection Tubes	25
Elution Tubes (1.7 mL)	25
Product Insert	1

Documentation

PROTOCOLS

(22550) Urine Exfoliated Cell and Bacteria RNA Purification Kit - Protocol (25 prep)

SAFETY DATA SHEETS

22550 - Urine Exfoliated Cell and Bacteria RNA Purification Kit - SDS

RELATED BLOGS

FAQs

Spin Column

Why do I have poor RNA recovery?

Poor RNA recovery could be due to one or more of the following:

Incomplete lysis of cells.
Ensure that the appropriate amount of Buffer SK was added to the exfoliated cell pellet. Ensure that the appropriate amount of lysozyme containing TE buffer and Buffer SK are added to the bacterial pellet in order to completely lyse the cells.
Column has become clogged.
Do not exceed the recommended amounts of 50 mL of urine or 1 x 10⁶ cells. The amount of starting material may need to be decreased if the column shows clogging below the recommended levels. See also “Clogged Column” below.
An alternative elution solution was used.
It is recommended that the Elution Solution E supplied with this kit be used for maximum RNA recovery.
Ethanol was not added to the lysate.
Ensure that the appropriate amount of ethanol is added to the lysate before binding to the column.
Ethanol was not added to the Wash Solution.
Ensure that 42 mL of 95% ethanol is added to the supplied Wash Solution A prior to use.
Low cell density in the sample
The cell number in different urine samples vary. While individuals with various diseases have >1000 exfoliated cells per mL of urine, a healthy male may have a number much lower than the 1000 cells per mL limit. It is possible that the total RNA isolated is not visible when resolved on an agarose gel or detected by spectrophotometry. In such cases, a larger input volume may be used. Alternatively, a more sensitive method such as BioAnalyzer or RT-PCR may be used for detection.
There is very little or no bacteria in the urine
The expected amount of bacteria in a urine sample is very little. A healthy individual usually has < 10,000 CFU/mL, therefore it is possible that the urine sample has very little bacteria present. The isolated RNA may not be visible when resolved on a gel. In such cases, a larger input volume may be used. Alternatively, a more sensitive method such as BioAnalyzer or PCR amplification may be used for detection.

I noticed the columns are getting clogged. What causes this?

Column clogging can result from the following:

Inefficient cell lysis.
Ensure that the appropriate amount of Buffer SK was added to the exfoliated cell pellet. Ensure that the appropriate amount of lysozyme containing TE buffer and Buffer SK are added to the bacterial pellet in order to completely lyse the cells.
Maximum number of cells exceeds kit specifications.
Do not exceed the recommended amounts of 50 mL of urine or 1 x 10⁶ cells.
Too many bacteria present in the urine.
The urine sample that was applied to the column contained too many bacterial cells. Reduce the amount of urine used. Clogging can be alleviated by increasing the g-force and/or centrifuging for a longer period of time until the urine passes through the column.
High amounts of genomic DNA present in sample.
The lysate may be passed through a 25 gauge needle attached to a syringe 5-10 times in order to shear the genomic DNA prior to loading onto the column.
Centrifuge temperature too low.
Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 20°C may cause precipitates to form that can cause the columns to clog.

Why is the RNA degraded?

RNA can be degraded due to following factors:

RNase contamination.
RNases may be introduced during the use of the kit. Ensure proper procedures are followed when working with RNA. Please refer to “Working with RNA” at the beginning of this user guide.
Procedure not performed quickly enough.
In order to maintain the integrity of the RNA, it is important that the procedure be performed quickly.
Lysozyme used may not be RNAse-free.
Ensure that the lysozyme being used with this kit is RNase-free, in order to prevent possible problems with RNA degradation.
Improper storage of the purified RNA.
For short term storage RNA samples may be stored at –20°C for a few days. It is recommended that samples be stored at –70°C for longer term storage.
The cells are old.
Older samples contain prematurely lysed cells which release RNase and can degrade RNA. Fresh urine samples are recommended.

Why is the purified RNA not performing well in downstream applications?

If the RNA does not perform well in downstream applications, It may be due to one or more of the following:

RNA was not washed twice with the provided Wash Solution A
Traces of salt from the binding step may remain in the sample if the column is not washed twice with Wash Solution A. Salt may interfere with downstream applications, and thus must be washed from the column.
There is ethanol carryover
Ensure that the dry spin under the Column Wash procedure is performed, in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.

I noticed genomic DNA contamination in the eluted RNA. How to prevent or correct this?

The contamination with genomic DNA may be due to the use of large amounts of starting material used. To address this issue, it’s encouraged to perform RNAse-free DNase I digestion on the RNA sample after elution to remove genomic DNA contamination. It is recommended that Norgen’s RNase-Free DNase I Kit (Cat. 25710) be used for this step.

Citations

Title	Development of a Voided Urine Assay for Detecting Prostate Cancer Noninvasively: A Pilot Study
Citation	BJU International 2017.
Authors	Trabulsi, E. J., Tripathi, S. K., Gomella, L., Solomides, C., Wickstrom, E., & Thakur, M. L.

Urine Exfoliated Cell and Bacteria RNA Purification Kit