Direct-TRI: High-throughput RNA-extracting Method for all Stages of Zebrafish Development

Materials and Reagents

1. 1.5 ml microcentrifuge tube (Rikaken, catalog number: RSV-MTT1.5)

2. Transfer pipet, 3 ml (Falcon, catalog number: 357575)

3. 8-strip tube, dome type (Rikaken, catalog number: RS-PCR-8D)

4. Cell culture dish, 100 mm (Nippon Genetics, catalog number: FG-2090)

5. Cell culture dish, 35 mm (Nippon Genetics, catalog number: TR4000)

6. Gemma Micro ZF 75 (Skretting)

7. Otohime B2 (Marubeni Nisshin Feed)

8. Kimwipe (Kimberly Clark)

9. AcroPrep Advance 96-well Long Tip Filter Plate for Nucleic Acid Binding (Pall, catalog number: 8133)

10. A reservoir for AcroPrep Advance (Merck, catalog number: BR701340-50EA)

11. Eppendorf twin.tec 96 well LoBind PCR plates (Eppendorf, catalog number: 0030129512)

12. DuraCross zebrafish breeding tank with divider (Laboratory Product Sales, catalog number: T233795)

13. Zebraifsh (Danio rerio)

14. TRI Reagent-LS (Molecular Research Center, catalog number: TS120)

    Notes:

    1. TRIzol-LS Reagent (Thermo Fisher Scientific, catalog number: 10296028) or ISOGEN-LS (Nippon Gene, catalog number: 311-02621) can also be used.

    2. Sepasol-RNA II Super (Nacalai Tesque, catalog number: 30487-46), an equivalent product, could not be used for RNA extraction from adult zebrafish because a precipitate was produced during the freezing and crushing processes.

15. Ethanol, 99.5% (Nacalai Tesque, catalog number: 08948-25)

16. Nuclease-free water (Merck, catalog number: H20MB1006)

17. BioMasher II (Nippi, catalog number: 320102; Figure 1)

    
    

    
    Figure 1. Image of BioMasher II




18. Liquid nitrogen

19. Pronase (Merck, catalog number: 53702)

20. QuantiFluor RNA System (Promega, catalog number: E3310)

21. Agilent RNA 6000 Pico kit (Agilent Technologies, catalog number: 5067-1513)

22. 10× Loading Buffer (Takara Bio, catalog number: 9157)

23. ExcelBand 1 KB (0.25-10 kb) DNA Ladder (Smobio, catalog number: DM3100)

24. Tricaine (Wako, catalog number: 051-06571)

25. Tris(hydroxymethyl)aminomethane (Rikaken, catalog number: RSP-THA500G)

26. Boric acid (Wako, catalog number: 021-02195)

27. EDTA-2Na (Wako, catalog number: 343-01861)

28. Agarose Powder (Rikaken, catalog number: RSV-AGRP-500G)

29. 10 mg/ml Ethidium bromide (Nippon Gene, catalog number: 315-90051)

30. Ethanol, 80% (see Recipes)

31. 1% agarose gel (see Recipes)

32. Tricaine (see Recipes)

33. E3 medium (see Recipes)

34. 20 mg/ml Pronase (see Recipes)

35. 5× TBE (see Recipes)

Equipment

1. Mortar and pestle, 120 mm (AsOne, catalog number: 6-549-03)

2. Vortex-Genie 2 (Scientific Industries)

3. PlateSpin II (Kubota)

4. Quantus Fluorometer (Promega, catalog number: E6150)

5. Agilent 2100 Bioanalyzer System (Agilent, catalog number: G2939B)

Procedure

1. Zebrafish maintenance and larvae collection

   1. Maintain adult zebrafish in a recirculation system at 28.5°C under 14:10 h light:dark cycle according to the standard protocol (Westerfield, 2000).

   2. Place female and male adult zebrafish in a breeding tank with a divider that separates fish to avoid immediate mating.

   3. Obtain fertilized eggs [embryos, 0 days post fertilization (dpf)] by removing the divider the next morning.

   4. Raise zebrafish embryos in E3 medium in a 100 mm cell culture dish at 28.5°C.

   5. At 1 dpf, transfer embryos with 2.5 ml of E3 medium into a 35 mm cell culture dish and add 150 μl of 20 mg/ml pronase.

   6. After 15 min incubation at room temperature (RT), dechorionate embryos by stirring the dish.

   7. Transfer embryos to a 100 mm cell culture dish with 20 ml of E3 medium and raise them up to 5 dpf at 28.5°C.

   8. Place zebrafish larvae (5 dpf larvae) in the recirculation system (30 larvae/tank).

   9. Feed zebrafish larvae in the recirculation system both paramecia and Gemma Micro ZF 75 twice a day from 5 to 30 dpf.

   10. Feed juveniles (30-90 dpf) and adults (~90 dpf) both brine shrimp and Otohime B2 twice a day after 30 dpf ( Wakamatsu et al., 2019 ).




2. RNA extraction from zebrafish larvae and juveniles

   Note: Use nuclease-free tips in all RNA extraction steps.

   1. Larvae (approximately 4-12 dpf, size smaller than 5 mm)

      1. Anesthetize a zebrafish larva in 20 ml of 0.2 mg/ml Tricaine at RT for 3 min.

      2. Prepare 100 μl of TRI Reagent-LS in 8-strip tube.

      3. Place a single zebrafish larva inside of a cap of the 8-strip tube and remove water using a micropipette (Figure 2).

         
         

         
         Figure 2. Zebrafish larvae placed in the tip of the 8-strip tube dome cap

      
      

      4. Assemble the cap to the 8-strip tube with each well containing the TRI Reagent, and lyse larvae completely by vortexing for 1 min at RT.

      5. Move to the RNA purification steps immediately or store the 100 μl lysates at -80°C for 1 month.

   2. Larva (approximately 14-38 dpf, size smaller than 10 mm)

      1. Anesthetize a zebrafish larva in 20 ml of 0.2 mg/ml Tricaine at RT for 3 min.

      2. Transfer a zebrafish larva in a 1.5 ml microcentrifuge tube with a Biomasher II and remove excess water using a micropipette.

      3. Add 50 μl of TRI Reagent-LS to the larva and immediately mash it at RT using a pestle until it is completely crushed.

      4. Add 50 μl of TRI Reagent-LS and vortex the sample for 1 min at RT.

      5. Move to the RNA purification steps immediately or store the 100 μl lysates at -80°C for 1 month.

   3. Juvenile (approximately 38 dpf, size larger than 10 mm)

      1. Anesthetize a zebrafish juvenile in 20 ml of 0.2 mg/ml Tricaine at RT for 3 min.

      2. Place a juvenile zebrafish in a 120 mm mortar and remove water by wiping the juvenile using Kimwipes.

      3. Pour liquid nitrogen to quickly freeze the juvenile.

      4. Grind the frozen juvenile into powder using a pestle (Figure 3).

         
         

         
         Figure 3. Image of a juvenile zebrafish on a mortar for grinding

      
      

      5. Add 1 ml of TRI Reagent-LS to the frozen powder and mix using a pestle until the mixture becomes liquid.

         Note: TRI Reagent-LS is frozen before the completion of tissue lysis. To lyse tissues completely, defrosting the regents is essential. Placing the mortar on a water bath (40-50°C) is sufficient to defrost the regents in a short time. The integrity of the RNA is not affected by this warming.

      6. Transfer 100 μl of the lysate to a new 1.5 ml microcentrifuge tube.

      7. Move to the RNA purification steps immediately or store the 100 μl lysates at -80°C for 1 month.

         Note: For reuse, the mortar should be washed with distilled water until the lysate is completely removed.

   4. RNA Purification

      1. Add 100 μl of 99.5% ethanol to the 100 μl lysate and mix by vortexing at RT.

         Note: When the lysate is stored in -80°C, thaw it at RT prior to this step.

      2. Prepare the AcroPrep Advance 96-Well Filter Plate on a reservoir (Figure 4).

         
         

         
         Figure 4. AcroPrep Advance 96-Well Filter Plate on a reservoir

      
      

      3. Transfer the 200 μl mixture into each well of the AcroPrep Advance plate.

      4. Centrifuge at 1,300 × g for 4 min at RT. Discard the flowthrough.

      5. Add 400 μl of 99.5% ethanol and centrifuge the plate at 1,300 × g for 4 min at RT. Discard the flowthrough.

      6. Repeat adding 400 μl of 99.5% ethanol and centrifuge the plate at 1,300 × g for 4 min at RT. Discard the flowthrough.

      7. Add 700 μl of 80% ethanol to the plate and centrifuge at 1,300 × g for 10 min at RT. Discard the flowthrough.

      8. Elute the RNA by adding 30 μl of nuclease-free water to the center of each well followed by incubation for 1 min at RT. Then, centrifuge at 1,300 × g for 4 min at RT. RNA solution can be stored at -80°C.




3. Determination of the RNA concentration

   Determine RNA concentration with the QuantiFluor RNA System kit and Quantus Fluorometer according to the manufacturer’s manual. Typical results are shown in Note 1.

   Note: The expected RNA concentration from an individual is too low to be measured with a spectrophotometer (e.g., NanoDrop).




4. RNA quality check

   We recommend checking the quality of RNA through agarose gel electrophoresis or with the Bioanalyzer for some but not all samples because the amounts of RNA are low in some cases.

   1. Agarose gel electrophoresis

      1. Add 15 μl (300 ng/lane) of the RNA sample into a well of 1% agarose gel.

      2. Run electrophoresis at 135 V for 25 min in 0.5× TBE. Typical results are shown in Note 2.

   2. Bioanalyzer

      This procedure requires the use of the Bioanalyzer.

      Check the RNA quality with the Agilent 2100 Bioanalyzer and the Agilent RNA 6000 Pico kit according to the manufacturer’s manual. Typical results are shown in Note 3.

Notes

1. Typical yields for RNA isolation with the Direct-TRI and phenol-chloroform are shown in Table 1. Owing to the limitation in the capacity of RNA to bind to the AcroPrep Advance 96 Filter Plate (approximately 3 μg), the yield of Direct-TRI tends to be smaller than that of the phenol-chloroform method ( Peterson et al., 2009 ) when adult zebrafish are used.

   
   

   Table 1. Total RNA amounts obtained with the Direct-TRI and phenol-chloroform methods
   

   	6 dpf (Vortex)	17 dpf (Biomasher)	84 dpf (Freezing homogenize)
   Direct-TRI	330 ng	3480 ng	2.8 μg
   Phenol-chloroform	170 ng	1500 ng	132 μg

   Note: RNA concentration was determined with the Quantus^TM Fluorometer.

   
   

2. A typical result of the RNA quality check through electrophoresis is shown in Figure 2. Zebrafish (6 dpf) were used to extract RNA using the TRI Reagent-LS to compare two different RNA-extracting methods, and then the RNA quality was checked. Two major bands (28S and 18S rRNA) indicate that RNA was not degraded (Figure 5). The small-size RNA was successfully obtained with the Direct-TRI method but not with the conventional phenol-chloroform method.

   
   

   
   Figure 5. Typical results of the RNA quality check through agarose electrophoresis




3. A typical result of the RNA quality check using the Bioanalyzer is shown in Figure 6. RNA was isolated from a 15 dpf larva with Direct-TRI, and the RNA quality was checked with the Bioanalyzer. Two RNA peaks (18S and 28S rRNA) were clearly observed, indicating that the RNA was not degraded (RIN 9.10). In the Direct-TRI method, the RIN value was approximately 7-9.5.

   
   

   
   Figure 6. Typical results of the RNA quality check with the Bioanalyzer

Recipes

1. E3 medium

   50× E3 stock solution

   NaCl 14.65 g (500 mM)

   KCl 0.63 g (17 mM)

   CaCl₂·2H₂O 2.43 g (33 mM)

   MgSO₄·7H₂O 4.07 g (33 mM)

   Distilled water mess up to 1 L

   Adjust pH to 7.2 with ~100 μl of 0.1 M NaOH

   Total 1 L

   Autoclave at 121°C for 20 min

   Store at RT

   
   

   E3 medium (1× E3 working solution)

   50× E3 stock solution 20 ml

   Distilled water mess up to 1 L

   Total 1 L

   Store at RT

2. 20 mg/ml Pronase

   Pronase 1 g

   Distilled water mess up to 50 ml

   Total 50 ml

   Divide into 1 ml aliquots

   Store at -20°C

3. Tricaine (Ethyl 3-aminobenzoate methanesulfonate)

   10 mg/ml Tricaine (Ethyl 3-aminobenzoate methanesulfonate)

   Tricaine 1 g

   Distilled water mess up to 100 ml

   Total 100 ml

   Divide into 1 ml aliquots

   Store at -20°C

   
   

   0.2 mg/ml Tricaine (Ethyl 3-aminobenzoate methanesulfonate)

   10 mg/ml Tricaine 1 ml

   Distilled water mess up to 50 ml

   Total 50 ml

4. 80% Ethanol

   Ethanol, 99.5% 40 ml

   Nuclease-free water 10 ml

   Total 50 ml

   Store at RT

5. 5× TBE

   Tris(hydroxymethyl)aminomethane 54.0 g

   Boric acid 27.5 g

   EDTA-2Na 1.85 g

   Distilled water mess up to 1 L

   Total 1 L

   Store at RT

6. 1% agarose gel

   5× TBE 35 ml

   Agarose Powder 3.5 g

   Distilled water 315 ml

   Total 350 ml

   Autoclave at 121°C for 1 min and cool to 50°C

   Add 17.5 μl of 10 mg/ml Ethidium bromide and pour into a gel plate

Acknowledgments

We thank the Hirata Laboratory members for fish care. This work was supported by the KAKENHI (Grant-in-Aid for Scientific Research B from the MEXT, Japan: 19H03329), the Takeda Science Foundation, and the Naito Foundation. This protocol is an updated version of the protocol used in our previous work (Maeta et al., 2020).

Competing interests

No competing interests declared.

Ethics

This study was approved by the Animal Care and Use Committee of the Aoyama Gakuin University (A9/2020) and conducted according to the Aoyama Gakuin University Animal Care and Use Guidelines and the Animal Research of In VIVO Experiments (ARRIVE) guidelines.

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