RNA Extraction Kit

RNA Extraction Kit

Desciption

This kit is optimized for use with 10 000 to 2 000 000 cells as starting material, providing 2 to 25 µg of total RNA. Use this kit in conjunction with ''MiniAmp mRNA'' amplification kit and with ''Amino Allyl'' labelling products and kits.
Starting samples can come from blood, cells or tissues.
This kit is suitable for preparation of total RNA in high yield and purity from a variety of tissues and cells samples. It can be used to purify RNA from tissues and cells, including plant, animal and yeast cells and protozoa.
The extracted RNA can be directly used either in Northern Blot and In vitro Translation techniques or as starting material for purification of mRNA for cDNA synthesis.
 
Reference Designation
REK Total RNA Extraction Kit (for 10 reactions)

Technical features & Protocol

Equipment needed

• 1.5 mL centrifuge tubes
• Ethanol (100%)
• Ice for incubations
• Micropipettes
• Vortex mixer
• Minicentrifuge

Kits contents

Items Packaging Storage
RBC Lysis Buffer 25ml in 30ml bottle -20°C Freezer
RNA Extraction Buffer 1.1ml in 2ml tube 20-25°C
RNA Wash Buffer Concentrate 0.5ml in 8ml bottle 20-25°C
RNA Micro Column 10 each 20-25°C
Elution Tube (1.5ml) 10 each 20-25°C
2 ml Wash Tube 10 each 20-25°C
Nuclease Free Water 1.5ml in 2ml tube -20oC Freezer

Total RNA extraction protocol from whole blood, cells or tissues

1. Spin all tubes 5 to 10 s before use.
2. Add 2 ml of 100 % ethanol to RNA Wash Buffer Concentrate before starting.
3. Add 200 µL of blood, cells or tissues to 2 mL of RBC Lysis buffer. Incubate on ice for 15 min. This step can be skipped when working with cells in culture.
4. Collect cell pellet by gentle centrifugation at 500 g for 5 min.
5. Remove and discard the supernatant.
6. Add 100 µL of RNA extraction buffer to the cell pellet. Resuspend gently by pipetting.
7. Homogenize the sample using a 1 mL disposable syringe with 20-gauge needle (5 to 8 times)
8. Spin 1 sec in a microcentrifuge at low speed. If a cell pellet forms, repeat homogenization. If not, proceed with the extraction.
9. Incubate on ice for 20 minutes.
10. Add 100 µl of 100% ethanol, mix briefly and incubate on ice for 10 minutes.
11. Load the entire contents onto an RNA Micro Column and place the column in a 2 ml wash tube.
12. Centrifuge for 1 minute at full speed in a microfuge (>10,000 rpm), discard the flow through and reuse wash tube..
13. Add 100 µl of RNA Wash Buffer to the column and centrifuge at full speed in a microfuge (>10,000 rpm) for 1 minute. Add another 100 µl of RNA Wash Buffer Concentrate to the column and centrifuge at full speed in a microfuge (>10,000 rpm) for 1 minute A longer centrifugation can be needed to remove completely the wash buffer.
14. Discard the 2 ml wash tube and carefully place the column in an elution tube.
15. Add 40 µl of nuclease-free water pre-warmed to 60°C into the center of the column.
16. Incubate 2 minutes and centrifuge at full speed in a microfuge (>10,000 rpm) for 1 minute.
17. Re-load the filtrate onto the column, incubate 2 minutes and centrifuge at full speed in a microfuge (>10,000 rpm) for 1 minute.
18. Discard the column and use the eluted total RNA for downstream applications.