RNAi reveals gene function due to the ability of the protocol to destroy the mRNA of the gene of interest resulting in a phenotype. The most challenging aspect of this experiment is the gene cloning and, for the lower level classes, the food preparation.
REQUIRED MATERIALS |
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TRIzol® |
Plastic tubes (15, 1.5 and 0.2 ml) and pestels |
Chloroform, Isopropanol and Ethanol |
Milli-Q water |
NanoDrop 1000 Spectrophotometer |
SuperScript III kit (Oligo(dT)20 primer, dNTPs Mix, 5x First strand buffer, DTT, RNase Inhibitor and SuperScript III retro transcriptase) |
Plastic tubes (0.2 ml) |
Thermocycler |
Primers F and R (Table 6) |
Phusion DNA polymerase, together with 5x High Fidelity buffer, dNTPs mix and MgCl2 |
Agarose and 1x TAE buffer |
Electrophoresis chamber |
Loading dye and 1 kb DNA ladder |
UV transilluminator |
QIAquick PCR Purification Kit or QIAquick Gel Extraction Kit (blade and thermoblock) |
Plasmid PR-T4P |
SmaI restriction enzyme and 10x NEB buffer #2 |
T4 polymerase, its buffer and BSA |
As for the WISH protocol, the first steps of the RNAi protocol in planarian are: look for mRNA species-specific sequences that correspond to the genes of interest and design F and R primers (Table 6) as described in Section 3.1, produce the cDNA and clone the gene of interest as described in Section 3.2, transform the bacteria and sequence the insert as described in Section 3.3.
REQUIRED MATERIALS |
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Ice and containers |
Blade and blender |
Food mill or sieve |
Spoons |
Small Petri dish (35 x 10 mm, 5 ml) or plastic wrap |
Fresh calf liver paste is prepared by trimming and homogenizing it in a blender. The liver should be kept on ice during the whole process to preserve it. The steps for the RNAi food preparation are:
REQUIRED MATERIALS |
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Option 2: Glycerol stock of transformed Escherichia coli HT115 stock |
2x YT medium-Kan (50 µg/ml)-Tet (12.5 µg/ml) broth |
Incubator at 37°C and shaker |
Plastic tube (1.5 and 50 ml) |
Spectrophotometer |
Isopropyl β-D-1-thiogalactopyranoside (IPTG) |
Calf liver paste (Section 4.2 and Red food dye |
Centrifuge and its adaptor for 50 ml tubes |
Instant Ocean® Sea Salt or 1x Montjuïc water |
Paper towel, Milli-Q water and ice |
The first RNAi method involves inducing bacteria transformed with the construct to transcribe the insert into the plasmid to generate double-stranded RNA (dsRNA) and then mixing these bacteria with the liver paste. When the planarians eat the food, planarians cells take up the dsRNA and it activate the pathway that degrades the mRNA with sequence complementary to one of its strands. This method works for Girardia sp. and D. dorotocephala, but P. morgani y P. gracilis do not eat food mixed with bacteria and the protocol reported in the Section 4.4 is necessary.
Thanks to the strong phenotype showed by the RNAi of the two selected genes (β-catenina-1 and ODF2), RNAi feedings are enough. Following this protocol, 250 ul of RNAi food (3-4 feedings for 30 to 50 worms) can be prepared from 50 ml transformed bacteria culture.
Keeping the construct (3 μl) on ice, 48 μl of competent Escherichia coli HT115 strain was added to it. The mix was incubated for 10 min on ice, and then a heat shock was performed for 35 sec at 42°C after which the tube was moved back into the ice. After 5 min, the transformed E. coli were placed at RT and 1 ml of Luria Broth (LB) without antibiotics were added. The cells were placed on a shaker at 250 rpm for 1 h at 37°C to recover and, finally, all the bacteria culture is moved into 4 ml of 2x YT medium-Kan (50 µg/ml)-Tet (12.5 µg/ml) broth culture.
Note: The glycerol stock is a mixture of 50% bacteria in broth culture and 50% glycerol that can be stored at -80°C for long time and used as starter for a new bacteria culture, when required. If a glycerol stock of bacteria HT115 already transformed with the construct of interest is available, few bacteria can be picked up from here and added to a 5 ml of 2x YT medium-Kan (50 µg/ml)-Tet (12.5 µg/ml).
Note: Cut the end of the tip for pipetting the liver.
Note: 1 ul of food/feeding/worm is usually required.
REQUIRED MATERIALS |
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Phusion DNA polymerase, together with GC buffer, primer T7, dNTPs mix and MgCl2 |
Thermocycler and plastic tube (0.2 and 1.5 ml) |
Agarose and 1x TAE buffer |
Electrophoresis chamber |
Loading dye and 1 kb DNA ladder |
UV transilluminator UV |
QIAquick PCR Purification Kit/td> |
T7 RNA polymerase together with its transcription buffer, NTPs mix (ATP, CTP, GTP and UTP), RNase Inhibitor |
Milli-Q water |
Thermoblock |
RQ1 DNase |
5 M Ammonia acetate |
70% and 100% Ethanol |
NanoDrop 1000 Spectrophotometer |
Calf liver paste (Section 4.2) and Red food dye |
The second method to induce RNAi is to mix the liver paste with purified dsRNA generated in vitro from the construct of interest. When the planarians eat the food, planarians cells take up the dsRNA and it activate the pathway that degrades the mRNA with sequence complementary to one of its strands. This method is best for P. morgani and P. gracilis, which do not eat food mixed with bacteria.
Once the gene of interest was successfully cloned, the insert was amplified by PCR reaction, using the construct as template, the primer T7 (5’-TAATACGACTCACTATAGGG-3’), the enzyme Phusion DNA polymerase and its GC buffer (New England Biolabs), following the manufacturer’s instructions. To obtain the required amount of amplified insert sequence, 240 ul (4 reaction/60 μl total each) of PCR reaction are prepared with 100 ng of template each reaction. The following touchdown PCR program was used:
A small aliquot of the amplified DNA (1-2 μl) will be run in 1% agarose gel in 1x TAE to check if the reaction worked properly. The DNA contained in the residual aliquot (237-238 μl) is purified with the QIAquick PCR Purification Kit.
The purified DNA was the template for the Transcription Reaction to produce the dsRNA. The reaction was done mixing 2.5-3 μg of the amplified and purified DNA fragment with the transcription buffer, the ribonucleotide mix (ATP, CTP, GTP and UTP), the T7 RNA polymerase (Invitrogen) and the RNase Inhibitor (Promega) in Milli-Q water to a final volume of 200 μl. Incubate the reaction ON at 37°C.
The dsRNA was treated with 5 μl RQ1 DNase (Promega). The reaction was incubated for 20 min at RT.
The dsRNA was precipitated following the protocol::
A small aliquot of the synthesized dsRNA (2 μl) will be run in 0.8% agarose gel in TAE to check if the reaction worked properly. It could happen that the band is not well defined or exactly at the expected size because of the different migration property of the dsRNA compared to the DNA and the RNA. The concentration of the dsRNA should be about 3-4 μg/ μl following the NanoDrop 1000 Spectrophotometer output. The dsRNA is stored at -80°C.
The day of the feeding, the dsRNA and one aliquot of liver paste are thawed. The dsRNA is mixed with the red food dye and then they are added to the liver paste and mixed together with the proportion liver:red food dye:dsRNA equal to 30:3:5.
REQUIRED MATERIALS |
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Petri dishes |
Instant Ocean® Sea Salt or 1x Montjuïc water |
Option 1: Calf liver paste mixed with bacteria (Section 4.2 and 4.3) |
Option 2: Calf liver paste (Section 4.2, Red food dye and dsRNA (Section 4.4) |
Squeeze bottle or standard bottle |
Waste container |
Paper towels or lab wiper KimWipes™ |
Optional for the amputation: Ethanol, Blade |
The suggested genes for the RNAi protocol have a really strong phenotype that appears after 3 feedings. For each experiment, 3-5 control worms and 3-5 RNAi worms, all of them 4-7 mm long and starved for at least 7-10 days were used.
Note: The food for Girardia sp. and D. dorotocephala is ready to use; the food for P. morgani and P. gracilis has to be prepared fresh, mixing the liver paste, the red food dye, and the dsRNA with the proportion 30:3:5.
Note: the phenotype for both β-catenin-1 and odf-2 gene emerge also in the worms that are not amputated but it takes more time and the efficiency is lower. If the researcher is interested in discovering the function of the gene of interest in the homeostasis of the worms they should not cut the worms and start the observation from the day after the last feeding. If the researcher is interest in discovering the function of the gene of interest in the regeneration of the worms they have to cut them and compare the fragment regeneration process between control and interfered worms.
Planarian species | Gene Name | Primer Sequence F | Primer Sequence R |
Sequence length |
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Girardia sp. | beta-catenin | TAGTATGTCAAGGCACAACG | GTATGGCCGATGTTAGTCTG | 2749 |
D. dorotocephala | beta-catenin | TTGGAAAGTCACAGTCACAG | GTGTCTAACATGCACACGTC | 2669 |
P. morgani | beta-catenin | GCCTATCGAACATAAGCATC | GTAGTCAATCGAAGGTCGAG | 3049 |
P. morgani | odf2 | TCAATTGCTCTAGCTGCTTC | AAAGGATCCAAGTCCACATT | 1003 |
P. gracilis | beta-catenin | TGGATTCAGAAGACCAGAAC | CGATCATGTGTGTTTGCTAC | 2613 |