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.
Remove excess H2O from embryos.
Using a 1mL syringe with a 20 gauge needle, add the appropriate amount (consult Table 1) of solution "D" to the embryos.
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.
Add 2M NaOAc pH4 (consult Table 1), and mix by inversion.
Add phenol and chloroform:isoamyl alcohol, vortex, and incubate on ice for 5-10'.
Centrifuge at 14,000 rpm for 2-3' at 4°C, and transfer upper aqueous phase to a new microcentrifuge tube.
Add 100% Isopropanol to precipitate RNA. Incubate samples at -20°C for at least 30'.
Centrifuge at 14,000 rpm for 10' at 4°C, and discard supernatant in waste container.
Dissolve the RNA pellet in solution "D", and transfer 10μL to a new microcentrifuge tube.
Precipitate RNA by adding equal volumes of 100% isopropanol. Incubate samples at -20°C for at least 30'.
Centrifuge at 14,000 rpm for 10' at 4°C, and discard supernatant in waste container.
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.
Centrifuge at 14,000 rpm for 10' at 4°C, and discard supernatant in waste container.
Pulse the pellet to collect remaining supernatant with a pipet tip. Allow the pellet to air dry or use a vacuum.
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.,
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.
