May 14, 2025
RNA extraction from Drosophila embryos
- Artem Ilin1
- 1SU
Protocol Citation: Artem Ilin 2025. RNA extraction from Drosophila embryos. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwq73dvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 14, 2025
Last Modified: May 14, 2025
Protocol Integer ID: 218238
Keywords: rna extraction from drosophila embryo, extraction from drosophila embryo, drosophila embryo, rna extraction, simple protocol for rna extraction, rna, cdna synthesis, embryo, extraction
Abstract
A simple protocol for RNA extraction and cDNA synthesis from Drosophila embryos
Materials
Invitrogen (Thermo Fischer)
TRIzol (cat number: 15596026)
Chloroform
Isopropanol
RNAse-free water
Sigma-Aldrich
DNAse I (AMPD1)
Applied Biosystems (Thermo Fischer)
High-Capacity RNA-to-cDNA Kit (Cat Number: 4387406)
Troubleshooting
RNA extraction
1h 3m 30s
Collect 100-200 staged embryos in a 1.5 ml eppendorf tube. Wash with PBT twice. Either centrifuge them at a low speed (<= 500 g) for a minute or let them precipitate by themselves.
10m
Remove all the liquid, add 50 µL of Trizol. Homogenize the embryos using a plastic pestle. Depending on the amount of material, add 50-150 µL of Trizol trying to wash the pestle. The protocol proceedes with the assumption that the total volume of Trizol is now 200 µL . Incubate for00:05:00 at room temperature.
10m
Add 1/5 volume of chloroform (for 200 µL of Trizol it would be 40 µL of chloroform). Vortex for 00:00:30 and incubate at RT for 00:03:00
3m 30s
Centrifuge for 00:15:00 at 12000 x g at 4 °C
15m
Transfer the aqueous phase to a new tube and add 0.8-1 volume of isopropanol. Incubate for 00:10:00 at RT if expected yield is high, or at -20 °C for 02:00:00 if you want RNA to precipitate better.
10m
Centrifuge for 00:10:00 at 12000 x g at 4 °C
10m
Discard the supernatant and wash the pellet one time with 200 µL of cold 70% EtOH. Try to dislodge the pellet from the tube side but do not suck it into the pipette tip when mixing.
Centrifuge for 00:05:00 at 10000 x g at 4 °C . Discard the supernatant.
5m
(Optional) Dry the pellet at 55 °C for 00:01:00
Resuspend the pellet in 10-20 µL of RNAse-free water.
(Optional) Dissolve at 55 °C for 00:01:00
Measure the RNA concentration at NanoDrop spectrophotometer.
DNAse treatment and reverse transcription
1h 35m
Use 1 µg of RNA from the previous step (the total volume shouldn't exceed 6.4 µL .
Add 0.8 µL of DNAse buffer and 0.8 µL of DNAse I from Sigma DNAse I Kit to your RNA, if the volume is less than 8 µL , add RNAse free water until it is equal to 8 µL . Mix by pipeting. Leave for 00:20:00 on RT. (Adding 1 µL of buffer and DNAse would not result in the failure of the experiment, so one can opt to do that, if their pipette is not very precise).
20m
Add1 µL of Stop solution, mix by pipeting and put on 65 °C for 00:10:00 .
10m
Transfer everything to a 200 μl PCR tube. Add 10 μl of RT buffer and 1 μl of RT enzyme from High-Capacity RNA-to-cDNA Kit. Mix by pipeting and put in the PCR machine for RT reaction. Set the program like this:
1. 37 °C for 01:00:00
2. 95 °C for 00:05:00
1h 5m
Add RNAse-free water to the final volume of 200 μl. Your cDNA is ready.