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Total RNA Isolation

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

# of embryos2550100
Solution "D" (µl)125250500
2M NaOAC pH 4 (µl)12.52550
Phenol, DEPC-H2O saturated (µL)125250500
49:1 Chloroform:Isoamyl alcohol (µL)2550100
100% Isopropanol (µL)125250500
Solution "D" (µL)50100150
100% Isopropanol (µL)40+1090+10140+10
75% EtOH (uL)40+1090+10140+10
DEPC-treated H20 or formamide (µL)10+522.5+547.5+5

Recipes

Denaturing Solution/Solution "D"

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