RNA Kit Instructions

Urine Extra-cellular RNA Isolation Kit Instructions

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Component

Contents

Storage

Treatment Buffer

10 mL

4C

Conditioning Buffer

4 mL

Room Temp

Precipitation Buffer

4 x 1 mL

-20C

Lysis Buffer

37.5 mL

-20C

Wash Buffer

15 mL

Room Temp

Elution Buffer

5 mL

Room Temp

Collection Tubes/Filter Columns

50

Room Temp

Elution Tubes

50

Room Temp

Storage Conditions and Product Stability

All solutions should be kept tightly sealed and stored at the indicated temperature. If any precipitation occurs in the Precipitation Buffer then heat to 95C for 5 minutes and invert. Repeat until the precipitate is mostly dissolved. Small residual precipitation will not affect performance. These reagents should remain stable for at least 1 year in their unopened containers.

Precautions and Disclaimers

This kit is designed for research purposes only. It is not intended for human or diagnostic use. Ensure that a suitable lab coat, disposable gloves, and protective goggles are worn when working with chemicals. Lysis Buffer contains guanidine thiocyanate and should be handled with care. Guanidine thiocyanate forms highly reactive compounds when combined with bleach, thus, care must be taken to properly dispose of any of this solution. If liquid containing these buffers is spilled, clean with suitable laboratory detergent and water. If the spilled liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.

Protocol

  1. Add 0.1 volume of Treatment Buffer to Whole Urine
    Example: for 1mL of urine add 0.1ml of Treatment Buffer; for 3ml of urine add 0.3ml Treatment Buffer
  2. Centrifuge at 1,000xg for 5min at RT. Transfer the supernatant to a new tube.
  3. Add 0.04 volume of Precipitation Reagent. Mix by inverting the tube 3 times.
    Example: for 1mL of urine add 0.04ml of Precipitation Reagent; for 3ml of urine add 0.12ml Precipitation Reagent
  4. Add 0.04 volume of Conditioning Buffer. Mix by inverting the tube 3 times.
    Example: for 1mL of urine add 0.04ml of Conditioning Buffer; for 3ml of urine add 0.12ml Conditioning Buffer
  5. Incubate on ice for 10min.
  6. Centrifuge at 3,000xg for 3min at RT. Discard the supernatant.
  7. Add 0.7ml of Lysis Buffer to the precipitate and pipette up and down until completely dissolved.
  8. Add 0.3ml of 100% isopropanol. Mix by inverting the tube 3 times.
  9. Place the Filter Column in the Collection Tube and add 0.5ml of the sample. Centrifuge at 500xg for 1min at RT. Discard the flow through. Repeat with remaining 0.5ml of the sample.
  10. Add 0.5ml of Wash Buffer. Centrifuge at 10,000xg for 15sec. Discard the flow through. Repeat 2 times (a total of 3 spins).
  11. Centrifuge at 10,000xg for 2min at RT.
  12. Place the Filter Column in a new Collection tube.
  13. Add 0.05ml of Elution Buffer to the Filter Column. Centrifuge 200xg for 2min at RT then 10,000xg for 2min.