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.
Table 1
# of embryos | 25 | 50 | 100 |
---|---|---|---|
Solution "D" (µl) | 125 | 250 | 500 |
2M NaOAC pH 4 (µl) | 12.5 | 25 | 50 |
Phenol, DEPC-H2O saturated (µL) | 125 | 250 | 500 |
49:1 Chloroform:Isoamyl alcohol (µL) | 25 | 50 | 100 |
100% Isopropanol (µL) | 125 | 250 | 500 |
Solution "D" (µL) | 50 | 100 | 150 |
100% Isopropanol (µL) | 40+10 | 90+10 | 140+10 |
75% EtOH (uL) | 40+10 | 90+10 | 140+10 |
DEPC-treated H20 or formamide (µL) | 10+5 | 22.5+5 | 47.5+5 |
Recipes
Denaturing Solution/Solution "D"
- 4 M Quanidine thiocyanate
- 25 mM Sodium Citrate
- 0.5% Sarkosyl
- 0.1 M Beta-Mercaptoethanol; add fresh.