Mike Barnkob:Protocols/Cloning/Isolating mRNA from spleen

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Isolating mRNA from spleen

Protocol on how to isolate mRNA from tissue, such as the spleen.

General notes

• A Mouse spleen is approximately 100-160 mg.
• The spin-column will be over-loaded if using too much materiale, which will result in low yield.

Reagents

For harvesting spleen:

1. 5 ml of R10 media in tube.
2. 5 ml 70% isopropanol in tube.
3. Keep both tubes on ice.

For homogenization:

1. 50 ml Falcon tube
2. 70μM cell strainer
3. R10 media
4. 2.5ml syringe
5. Red blood cell lysis buffer

For mRNA isolation:

1. Qiagen RNeasy Mini Kit
2. 70% ethanol

Protocol

Harvest spleen:

1. Euthanize mouse by cervical dislocation.
2. Dissect out spleen
   Note: Spray hair with isopropanol.
   Note: Keep hair out of incision.
3. Using forceps, dip spleen into isopropanol tube and leave for 30 seconds.
4. Transfer spleen into RPMI.
5. In tissue culture hood, cut spleen into smaller pieces (4-5 in total)

Homogenize tissue and lyse red blood cells:

1. Work in tissue culture hood
2. Add 1mL of R10 to 50 ml Falcon tube. Place 70μM cell strainer on tube and add part of spleen on top of this.
   Note: The mRNA isolation kit can maximum handle 30 mg of tissue. Aim for 10-20 mg per part.
   Note: Left-over spleen can be kept in RNALater media from Qiagen.
3. Take out plunger of 2.5ml syringe, and mesh spleen through strainer with blunt end of the plunger.
   Note: Flush cells through with a little R10.
   Note: When done, add up to 5 ml R10.
4. Spin cells down at 1500 RPM for 5 min and remove supernatant.
5. Resuspend in 2 ml of red blood cell lysis buffer and leave on ice for 5 min.
6. Add 5 ml of R10, spin cells down at 1500 RPM for 5 min and remove supernatant.
7. Resuspend in 1 ml of R10.
   Optional: count cells.

mRNA isolation:

Follows Qiagen RNeasy Mini Kit instructions and presumes all reagents have been prepared.

1. Spin cells down at 1500 RPM for 5 min and remove supernatant.
2. Disrupt and homogenize:
   • Resuspend in 600μL buffer RLT and pipet up and down several times.
   • Transfer to 1.5 Eppendorf tube
   • Centrifuge the lysate for 3 min at full speed and transfer the supernatant to new 1.5 Eppendorf tube.
     Note: The lysate is normally between 250-600 μL.
   • Add 1 volume of 70% ethanol to the lysate, and mix immediately by pipetting (don't vortex).
3. Transfer 700 μL of sample to an RNeasy spin column placed in a 2 ml collection tube. Close lid gently, and centrifuge for 15 s at 10,000 rpm. Discard the flow-through.
4. Add 700 μL Buffer RW1 to the RNeasy spin column. Close lid gently, and centrifuge for 15 s at 10,000 rpm. Discard the flow-through.
5. Add 500 μL Buffer RPE to the RNeasy spin column. Close lid gently, and centrifuge for 15 s at 10,000 rpm. Discard the flow-through.
6. Add 500 μl Buffer RPE to the RNeasy spin column. Close lid gently, and centrifuge for 2 minutes at 10,000 rpm. Discard the flow-through.
7. Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30–50 μL RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at 10,000 rpm to elute the RNA.
8. Label and store at -20°C.

Quality control: Using a NanoDrop

1. A260/A280: ratio should be close to 1.8-2.0 [1].
2. A260/A230: ratio should be close to 2.0 when isolating low amounts of RNA. Values < 1.0 indicates contamination with salts or rests of phenol or protein in the RNA solution.

Storage:

• Purified RNA may be stored at –20°C or –70°C in RNase-free water. Under these conditions, no degradation of RNA is detectable after 1 year.

References

• RNeasy Mini Handbook, p37: http://www.qiagen.com/gb/resources/resourcedetail?id=14e7cf6e-521a-4cf7-8cbc-bf9f6fa33e24&lang=en
• RNA quality control: http://biomedicalgenomics.org/RNA_quality_control.html