RNA extraction from individual zebrafish embryos

Total nucleic acid extraction from single zebrafish embryos for genotyping and RNA-seq

Clean bench, equipment and gloves with RNAzap

1. Nucleic acid extraction

Take the Agencourt RNA clean XP beads (Beckmann Coulter, Cat # A63987) out of the fridge. They need to come to room temperature for 30 mins before use. Shake well.

AMPure XP Beads (Beckman Coulter, Cat # A63881) also work.

Prepare fresh 70% ethanol. You will need ~700 µl per well.

• Collect single embryos in each well of 2 ml round well round bottom assay blocks (Axygen Cat # P-DW-20-C-S). Collect the embryos in 9 µl of egg water using truncated pipette tips. Label the plate with well numbers that the embryos are collected in and the experiment name on the front and the side. Seal the plate using adhesive plate seals (Thermo Fisher Scientific, Cat # AB0558 or 3M sealer). Pull the seal on the sides and secure the plate. Do not pull them over the labelled side as the ink may come off when the seal is removed. Immediately place on dry ice for ~ 5 mins. From this step, you can either proceed to extraction straight away or store the plate at -80 till further needed.

• RLT buffer (Qiagen, Cat # 79216) (as needed) (stored at RT) + 10 µl of 14.3M beta mercaptoethanol  (Sigma Aldrich, Cat # M3148-100 ml) for every 1 ml of RLT buffer. For single embryo extractions, 110 µl of RLT buffer/well is enough to sufficiently lyse the tissue. Make a pre-mix in a 5 ml tube for up to 24 samples or multi-channel dispensing dish. Put all waste plastic in correct fume hood waste jar.

On the day of extraction

• Remove Agencourt RNA clean XP beads from fridge to come to room temperature over 30 minutes. Shake well.

Under the fume hood 

• Prepare lysis buffer as below –
• RLT buffer (Qiagen, Cat # 79216) (as needed) (stored at RT) + 10 µl of 14.3M beta mercaptoethanol  (Sigma Aldrich, Cat # M3148-100 ml) for every 1 ml of RLT buffer. For single embryo extractions, 110 µl of RLT buffer/well is enough to sufficiently lyse the tissue. Make a pre-mix in a 5 ml tube for up to 24 samples or multi-channel dispensing dish. Put all waste plastic in correct fume hood waste jar.

If you are using RLT plus buffer add 0.5% v/v of Reagent Dx. Buffer RLT plus contains detergent which foams on agitation. Reagent Dx is an antifoaming reagent and prevent loss of material as foam.

• Take the embryo plate out of -80 on ice. Can be stored in the fridge briefly. While still on ice add one 5mm stainless steel beads (Qiagen, Cat # 69989) in each well of the plate containing the embryos with clean gloved hands. Then add 110 µl of RLT buffer containing BME to the wells using a multichannel. Act quickly; the embryos should not thaw without the buffer as this causes degradation of sample. 

• Seal the plate 6 times using a heat seal at 170⁰C for 3 seconds. Wait a few seconds between seals and secure the sides by using a plate seal roller (Bio-Rad Laboratories, Cat # MSR0001). The extra layers of seals ensure that the bead does not pierce the seal during agitation.

• Arrange the plate in the slots of the tissue lyser adaptor (Qiagen, Cat # 85300) (Read manual for more information). Make sure both the adaptors are balanced. To fit the plate in the tissue lyser, remove the tube adaptor and replace with the sealed 2 ml assay block.

• Adjust the time to 5 mins and frequency to 12 Hz using the dials on the machine. 

Note – Check that the plate is aligned properly between the adaptors and is not at an angle.

• Close the hood and press start. If the machine wobbles during lysis that often means that it is not balanced. In such a situation, stop the machine, rearrange the plate and start again.

• While the sample is lysing, add 1.8x AgenCourt RNA Clean XP beads ~180 µl (if you have lysed in 110 µl) to a clean V bottom 0.3 ml plate (AB1400L)

• Once the tissue lyser stops, take the plate out and spin briefly to collect the lysed sample at the bottom. Examine the lysate, if it does not appear homogenous, lyse again for 2 or more mins till the lysate appears homogenous.

Note – The following steps need to be done under the fume hood.

• Add the lysate to 1.8x AgenCourt RNA Clean XP beads in the V bottom 0.3  ml plate made 2 steps above and mix well by pipetting up and down using a multichannel.

• While the samples are incubating, label a V bottom 0.3 ml plate (AB1400L). This is for the supernatant that is removed in the next steps

• Incubate in the fume hood at room temperature for 15 minutes.
• Place the plate containing the sample on the magnet (DynaMag™-96 Side Skirted Magnet, Cat # 12027) and leave for 10 minutes or until the solution is clear.
• Remove supernatant using a pipette (without disturbing beads) and transfer to a fresh clearly labelled plate. (Retain until samples have passed)
• While still on the magnet, add 200 µl of 70% ethanol (made from standard stock ethanol) to cover beads without disturbing and leave for 30 seconds. Do not resuspend the beads.
• Carefully remove ethanol without disturbing the beads into the ethanol waste and repeat wash for a total of 3 times.
• In the final wash, thoroughly remove any residual ethanol using a multichannel without disturbing the beads.
• Air dry sample at room temperature for 10-15 minutes or until there is no remaining trace of liquid and the beads appear dry. Do not over dry, else re-suspending the beads becomes difficult.
• Add 58 µl RNAse free water and re-suspend beads using pipette. DO NOT VORTEX. Using the same tip gently flush the beads down the tube by pipetting the water over the beads: if necessary, manipulate beads down the tube using the pipette tip.

Note – If more concentrated sample is desired, a smaller volume of water can be used. Use at least 35 µl of water to ensure optimum recovery.

• Incubate on the bench at room temperature for 5 minutes.
• Place on magnet and leave for 5 minutes or until the solution is clear.
• Carefully transfer supernatant to a fresh clearly labelled 96 well superplate (Thermo Fisher Scientific, AB-2800) being careful not to take up any beads.

2. Genotyping

Genotype for each embryo is determined using KASP genotyping.

For KASPar genotyping, use the following –

100x master mix

KASPar Rxn mix – 400 µl (2x RM in freezer)

MgCl2 – 6.4 µl

Water – 400 µl

Assay Mix – 11 µl

Add 8 µl of master mix per well in a 96 well KASPar plate (Thermo Scientific, Cat # AB-0800/K). 

Add 1ul of extracted sample as template per well.  This is less DNA volume than HotShot and therefore there is more water in the master mix. 

Run using the below program-

1. 94 ° - 15 mins
2. 94 ° - 10 secs
3. 57 ° - 5 secs
4. 72 ° - 10 secs
5. Go to 2, 20 times
6. 94 ° - 10 secs
7. 57 ° - 20 secs
8. 72 ° - 40 secs
9. Go to 6, 18 times
10. 10 ° forever

3. RNA Quantification

• Measure RNA concentration using Quant-iT™ RNA Assay Kit (Invitrogen, Q33140) as per manufacturer’s instructions.
• Calculate volume of RNA needed for the experiment. In a separate 96 well plate, transfer the appropriate volume of RNA. Use the same concentration for all samples.

4. DNAse treatment

• Add 1ul of DNAseI (New England Biolabs, Cat # M0303L) and 10 µl of 10x DNAseI buffer (Thermo Scientific, Cat # AM8170G) to each well. We need extra buffer which is ordered separately.
• Make up the total volume to 100 µl with RNAse free water i.e. RNA + DNAseI + 10x DNAseI buffer + RNAse free water = 100 µl/well.
• Seal the plate and heat at 37° for 10 minutes. 
• Add 1ul of 0.5M EDTA to each well (Pre-made solution Sigma E7889).
• Heat at 75° for 10 minutes.

• Transfer DNAsed sample to a V bottom 0.3 ml plate. Add 200 µl of Agencourt RNA XP beads to each well and mix well by pipetting.

• Incubate at room temperature for 15 minutes

• Place the plate containing the sample on the plate magnet and leave for 10 minutes or until the solution is clear.
• Remove supernatant using a pipette (without disturbing beads) and transfer to a fresh clearly labelled plate. (Retain until samples have passed)
• While still on the magnet, add 200 µl of 70% ethanol to cover beads without disturbing and leave for 30 seconds. Do not resuspend the beads.
• Carefully remove ethanol without disturbing the beads and repeat wash for a total of 3 times.
• In the final wash, thoroughly remove any residual ethanol using a multichannel without disturbing the beads.
• Air dry sample at room temperature for 10-15 minutes or until there is no remaining trace of liquid and the beads appear dry. Do not over dry, else re-suspending the beads becomes difficult.
• Add 52 µl RNAse free water and re-suspend beads using pipette. DO NOT VORTEX. Using the same tip gently flush the beads down the tube by pipetting the water over the beads: if necessary, manipulate beads down the tube using the pipette tip.

Note – This elution volume is for TruSeq Stranded Illumina kits. Please adjust the elution volume to match the first step in the protocol that you will be using.

• Incubate on the bench at room temperature for 5 minutes.
• Place on magnet and leave for 5 minutes or until the solution is clear.
• Carefully transfer 50 µl of supernatant to a fresh clearly labelled 96 well superplate being careful not to take up any beads.
• Proceed to fragmentation and library prep.