Urine Total RNA Purification Maxi Kit (Slurry Format)

Features and Benefits

Isolate high quality total RNA and all sizes of circulating and exosomal RNA, including microRNA
No phenol extractions
Very sensitive and linear without the need for carrier RNA
Bind and elute all RNA irrespective of size or GC content, without bias
Isolate inhibitor-free urinary RNA
Slurry/Spin column and Slurry/96-well format available
Purification is based on Norgen’s proprietary resin matrix

Norgen’s Urine Total RNA Purification Maxi Kit (Slurry Format) provides a rapid method for the isolation and purification of total RNA from 5 – 10 mL of urine. The kit purifies all sizes of RNA, from large mRNA and ribosomal RNA down to small RNAs. These small RNAs include regulatory RNA molecules such as microRNA (miRNA) as well as tRNA and 5S rRNA. Small RNA molecules are often studied due to their ability to regulate gene expression. miRNAs are typically 20-25 nucleotides long, and regulate gene expression by binding to mRNA molecules and affecting their stability or translation. Several recent studies showed that miRNA regulates cell growth and apoptosis. Furthermore, clinical and experimental analyses suggested that miRNAs may function as a novel class of oncogenes or tumor suppressor genes. MicroRNA expression profiles of different tumor types, relative to their normal tissues, have recently been shown to provide phenotypic signatures for particular cancer types. Unique patterns of aberrant miRNA expression may serve as molecular biomarkers for tumor diagnosis, prognosis of disease specific outcomes, and prediction of therapeutic responses.

Details

Supporting Data

Figure 1. Isolation and Detection of Total RNA from 10 mL Urine Samples. Norgen's Urine Total RNA Purification Maxi Kit (Slurry Format) was used to isolate total RNA from two 10 mL urine samples. The purified total RNA was then used as the template in an RT-qPCR reaction to detect the human miR-21 gene (Red line) and the human 5S gene (Blue line). Five microlitres of the isolated RNA was used as the template in the RT step, and 5 µL from the RT step was used in the qPCR reaction. As it can be seen, the qPCR was able to successfully detect and amplify both the 5S gene and the miR-21 gene in all cases, indicating the high quality of the isolated urine total RNA. The black line in the graph above corresponds to the No Template Control.

Kit Specifications
Volume of Urine Processed	5 - 10 mL
Size of RNA Purified	All sizes, including < 200 nt
Time to Complete 10 Purifications	< 30 minutes
Average Yield	Variable depending on specimen

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. 29600 (variable)	Cat. 29650 (96 preps)
Slurry C3	20 mL	2 x 24 mL
Lysis Buffer A	2 x 130 mL	4 x 130 mL
Wash Solution A	38 mL	2 x 20 mL
Elution Solution A	6 mL	2 x 6 mL
96-Well Filter Plate	-	1
Adhesive Tape	-	1
Mini Filter Spin Columns	50	-
Collection Tubes	50	-
96-Well Collection Plate	-	1
Elution Tubes (1.7 mL)	50	-
96-Well Elution Plate	-	1
Product Insert	1	1

Documentation

RELATED BLOGS

FAQs

Maxi Slurry, High Throughput Maxi Slurry

If I am not going to process my samples immediately, how should I store my samples?

We recommend the use of Norgen’s Urine Preservative, which is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures. The components of the Urine Preservative allow samples to be stored for over 2 years at room temperature with no detected degradation of urine DNA, RNA or proteins. Norgen’s Urine Preservative is available in 2 convenient formats: in a liquid format in Norgen’s Urine Preservative Single Dose Ampules, as well as in a dried format in Norgen’s Urine Collection and Preservation Tubes.

What If a variable speed centrifuge is not available?

A fixed speed centrifuge can be used, however reduced yields may be observed.

What will happen if my centrifugation speed varied from the recommended speed?

This may decrease the binding of the RNA to the column.

At what temperature should I centrifuge my samples?

All centrifugation steps are performed at room temperature. Centrifugation at 4°C will not adversely affect kit performance.

My centrifuge speeds are defined in rpm and not in g-force. How can I convert g-force to rpm?

The correct rpm can be calculated using the formula:

RPM= √RCF/(1.118x10^-5)(r)

Where RCF = required gravitational acceleration (relative centrifugal force in units of g); r = radius of the rotor in cm; and RPM = the number of revolutions per minute required to achieve the necessary g-force.

Can I process a different urine volume?

Yes, you can. To process different urine volumes (up to 5mL) please check Table 1 in the protocol for the appropriate volumes of Lysis Buffer A and 96-100% Ethanol to be added to different urine sample volumes. The volume of Slurry C3 is fixed for all urine volumes. The minimum recommended urine input is 3 mL, and the maximum recommended input is 5 mL.

What if I added more or less from the specified reagents’ volume?

Adding less volume may reduce your RNA yields. Adding more may not affect the RNA yields EXCEPT if more Elution Solution A was added. Eluting RNA in a higher volume of Elution Solution A will result in diluting your RNA.

What if I forgot to do a dry spin after my third wash?

Your RNA elution will be contaminated with traces of the Wash Solution A. This may dilute the RNA yield in elution. Also, it may interfere with your downstream applications. Re-isolate the eluted RNA using the same procedure as you initially isolated the RNA from urine but using your elution as your input.

Why did my samples show very low RNA yields?

Some urine samples contain very little RNA. This varies from individual to individual based on numerous variables. In order to increase the yield, the amount of urine input could be increased.

Why does my RNA not perform well in downstream applications?

If a different Elution solution was used other than the one provided in the kit, the buffer should be checked for any components that may interfere with the application. Common components that are known to interfere are high salts (including EDTA), detergents and other denaturants. Check the compatibility of your elution buffer with the intended use.

What if the solutions did not flow through the column?

The centrifugation speed was too low. Check the centrifuge to ensure that it is capable of generating a sufficient centrifugal force that is required to move the liquid phase through the resin. You may also spin an additional two minutes to ensure that the liquid is able to flow completely through the column.

Why is the 96-Well Plate was clogged during vacuuming?

This indicates an insufficient vacuum. Ensure that a vacuum pressure of at least -650 mbar or -25 inHg is developed.

Can I use frozen Urine samples for isolating miRNA?

It is recommended that the Urine samples should be centrifuged to prepare cell-free samples before freezing them.

We recommend the following steps to prepare frozen urine for isolation:

Gently warm the sample to room temperature or 37°C for 5 min.
DO NOT perform a centrifugation step after thawing frozen cell-free Urine samples - this will eliminate the precipitated proteins leading to loss of protein-bound cf-NA or exosomes.
Proceed with the protocol.

We recommend the use of Norgen’s Urine Preservative when collecting urine samples. Norgen’s Urine Preservative is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures, therefore no protein precipitation will occur and the purified nucleic acids will be of a higher quality.

Citations

Title	Emerging Utility of Urinary Cell-free Nucleic Acid Biomarkers for Prostate, Bladder, and Renal Cancers
Citation	European Urology Focus 2017.
Authors	Lin, S. Y., Linehan, J. A., Wilson, T. G., & Hoon, D. S. (2017)

Title	High-throughput miRNA sequencing and identification of biomarkers for forensically relevant biological fluids
Citation	Electrophoresis 2016.
Authors	Seashols-Williams, S., Lewis, C., Calloway, C., Peace, N., Harrison, A., Hayes-Nash, C., ... & Zehner, Z. E.

Urine Total RNA Purification Maxi Kit (Slurry Format)