Urine Exosome and Free-Circulating RNA Isolation Kits

Supporting Data

Figure 1. Isolation of RNA from exosomes purified from different urine volumes. Norgen’s Urine Exosome and Free-Circulating RNA Isolation Mini Kit (Cat# 59200) was used to isolate exosomal RNA from different urine volumes ranging from 0.25 mL and up to 1 mL. Two microlitres of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated urinary exosomal miR-30a. (A) The urinary exosomal miR-30a is linearly decreasing with increasing the sample input volume. B) The relative amount of the urinary exosomal miR-30a shows excellent linearity with a percentage of recovery of more than 90%.

Figure 2. Isolation of Free-Circulating RNA after the isolation of Exosomal RNA from different urine volumes. Norgen’s Urine Exosome and Free-Circulating RNA Isolation Mini Kit (Cat# 59200) was used to isolate Free-Circulating RNA after the isolation of exosomal RNA from different urine volumes ranging from 0.25 mL and up to 1 mL. Two microlitres of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated urinary free-circulating miR-21. (A) The free-circulating miR-21 is linearly decreasing with increasing the sample input volume. B) The relative amount of the free-circulating miR-21 shows excellent linearity with a percentage of recovery of more than 90%.

Figure 3. Isolation of RNA from exosomes purified from different urine volumes. Norgen’s Urine Exosome and Free-Circulating RNA Isolation Midi Kit (Cat# 59300) was used to isolate Exosomal RNA from different urine volumes ranging from 2 mL and up to 10 mL. Two microlitres of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated urinary exosomal miR-30a. (A) The urinary exosomal miR-30a is linearly decreasing with increasing the sample input volume. B) The relative amount of the urinary exosomal miR-30a shows excellent linearity with a percentage of recovery of more than 90%.

Figure 4. Isolation of Free-Circulating RNA after the isolation of Exosomal RNA from different urine volumes. Norgen’s Urine Exosome and Free-Circulating RNA Isolation Midi Kit (Cat# 59300) was used to isolate Free-Circulating RNA from after the isolation of Exosomal RNA from different urine volumes ranging from 2 mL and up to 10 mL. Two microlitres of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated urinary free-circulating miR-21. (A) The free-circulating miR-21 is linearly decreasing with increasing the sample input volume. B) The relative amount of the free-circulating miR-21 shows excellent linearity with a percentage of recovery of more than 90%.

Figure 5. Isolation of RNA from exosomes purified from different urine volumes. Norgen’s Urine Exosome and Free-Circulating RNA Isolation Maxi Kit (Cat# 59400) was used to isolate exosomal RNA from different urine volumes ranging from 11 mL to 30 mL. Two microlitres of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated urinary exosomal miR-30a. (A) The urinary exosomal miR-30a is linearly decreasing with increasing the sample input volume. B) The relative amount of the urinary exosomal miR-30a shows excellent linearity with a percentage of recovery of more than 90%.

Figure 6. Isolation of Free-Circulating RNA after the isolation of Exosomal RNA from different urine volumes. Norgen’s Urine Exosome and Free-Circulating RNA Isolation Maxi Kit (Cat# 59400) was used to isolate Free-Circulating RNA after the isolation of exosomal RNA from different urine volumes ranging from 11 mL to 30 mL. Two microlitres of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated urinary free-circulating miR-21. (A) The free-circulating miR-21 is linearly decreasing with increasing the sample input volume. B) The relative amount of the free-circulating miR-21 shows excellent linearity with a percentage of recovery of more than 90%.

Kit Specifications
Minimum Urine Input	250 µL
Maximum Urine Input	1 mL
Size of RNA Purified	All sizes, including miRNA and small RNA (< 200 nt)
Elution Volume	50 - 100 µL
Time to Complete 10 Purifications	35 - 40 minutes
Average Yields*	Variable depending on specimen

*Please check page 4 of the product insert for the average yields and the common RNA quantification methods.

Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.

Important Note
Urine samples stored at -70°C, -20°C or at 4°C will develop some precipitation due to the aggregation of some of the highly abundant proteins in urine. Eliminating these precipitates using centrifugation or filtration may cause the loss of exosomes. Furthermore, these precipitates may affect the quality of the purified nucleic acid. We recommend the use of Norgen’s Urine Preservative when collecting urine samples, 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 as 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.

Component	Cat. 59200 (50 preps)	Cat. 59300 (25 preps)	Cat. 59400 (15 preps)
Slurry E	12.5 mL	12.5 mL	12.5 mL
ExoC Buffer	8 mL	30 mL	50 mL
ExoR Buffer	12 mL	12 mL	12 mL
Lysis Buffer A	2 x 20 mL	2 x 20 mL	2 x 20 mL
Lysis Additive B	2 mL	2 mL	2 mL
Wash Solution A	2 x 18 mL	18 mL	18 mL
Elution Solution A	2 x 6 mL	6 mL	6 mL
Mini Filter Spin Columns	50	25	15
Mini Spin Columns	100	50	30
Collection Tubes	100	50	30
Elution Tubes (1.7 mL)	100	50	30
Product Insert	1	1	1