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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
