Hackett Lab

Guanidine-based isolation

Objective:

To obtain total RNA from zebrafish embryos.

Required Materials

• Denaturing Solution or Solution "D"
• 2 M NaOAC pH 4
• Phenol, H₂O saturated
• 49:1 Chloroform/Isoamyl alcohol
• Isoproponal
• 75% EtOH
• DEPC-treated H₂O or freshly deionized formamide
• 1 mL syringe
• 20 gauge needle
• 1.5mL microcentrifuge tubes
• microcentrifuge

Procedures

Start=> Collected zebrafish embryos of desired developmental stage, etc.

1. Remove excess H₂O from embryos.
2. Using a 1mL syringe with a 20 gauge needle, add the appropriate amount (consult Table 1) of solution "D" to the embryos.
3. Homogenize the embryos by passage through the syringe needle (5-8) times. To decrease the volume of frothy homogenate and check the status of the embryos, briefly pulse in a microcentrifuge at low speed. If there is a pellet of embryonic tissue, continue to homogenate with the syringe needle. If not, continue with the extraction.
   • The homogenates can be safely stored at -80°C.
4. Add 2M NaOAc pH4 (consult Table 1), and mix by inversion.
5. Add phenol and chloroform:isoamyl alcohol, vortex, and incubate on ice for 5-10'.
6. Centrifuge at 14,000 rpm for 2-3' at 4°C, and transfer upper aqueous phase to a new microcentrifuge tube.
7. Add 100% Isopropanol to precipitate RNA. Incubate samples at -20°C for at least 30'.
8. Centrifuge at 14,000 rpm for 10' at 4°C, and discard supernatant in waste container.
9. Dissolve the RNA pellet in solution "D", and transfer 10µL to a new microcentrifuge tube.
10. Precipitate RNA by adding equal volumes of 100% isopropanol. Incubate samples at -20°C for at least 30'.
11. Centrifuge at 14,000 rpm for 10' at 4°C, and discard supernatant in waste container.
12. Wash pellet by adding 75% EtOH.
    • If RNA is to be stored, leave majority of RNA precipitated in the 75% EtOH at -80°C. Continue with the 10µL aliquot to determine approximate concentration and integrity of rRNA bands.
13. Centrifuge at 14,000 rpm for 10' at 4°C, and discard supernatant in waste container.
14. Pulse the pellet to collect remaining supernatant with a pipet tip. Allow the pellet to air dry or use a vacuum.
15. Resuspend in DEPC-treated H₂O or formamide.
    • Suspension in formamide protects the RNA from degradation by RNases, but for most applications the RNA should be precipitated from the formamide by adding 4 volumes of 100% EtOH and centrifuging at 14,000 rpm for 10' at 4°C.,
16. Quantitate RNA by diluting 1 µL into 100µL with alkaline H₂O (see below). Then determine the A₂₆₀ and A₂₈₀.
    • Water used for spectrophotometric measurement of RNA should have a pH of > 7.5. Acidic pH affects the UV absorption spectrum of RNA and significantly decreases the A₂₆₀/A₂₈₀. Adjust water to a slightly alkaline pH by adding concentrated Na₂HPO₄ solution to a final concentration of 1mM.

Table 1

Recipes

Denaturing Solution/Solution "D"

• 4 M Quanidine thiocyanate
• 25 mM Sodium Citrate
• 0.5% Sarkosyl
• 0.1 M Beta-Mercaptoethanol; add fresh.