Jun 10, 2026
qRT-PCR for Zebrafish Brian Tissue
- Robin Zhang1
- 1UCL
- Tuschl lab
Protocol Citation: Robin Zhang 2026. qRT-PCR for Zebrafish Brian Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lymzbelx9/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: June 02, 2026
Last Modified: June 10, 2026
Protocol Integer ID: 318395
Keywords: pcr for zebrafish brian tissue, zebrafish brian tissue, sample preparation to qpcr, qpcr, pcr, sample preparation, qrt
Abstract
From sample preparation to qPCR
Safety warnings
Tissue collection and dissection must not be performed unless the user has confirmed and complied with all applicable institutional, national, and local animal research regulations. Depending on the animal species, zebrafish developmental stage or age, tissue type, and local requirements, prior approval from an IACUC, equivalent ethics committee, animal welfare body, or regulatory authority may be required. Users are responsible for obtaining the appropriate approval, licence, or institutional permission before carrying out any tissue collection.
Ethics statement
This protocol involves the collection and dissection of animal tissue and must be carried out strictly in accordance with internationally accepted standards for animal research, as well as all applicable institutional, national, and local regulations. Before using this protocol, users must determine whether prior ethical approval is required from an Institutional Animal Care and Use Committee (IACUC), equivalent ethics committee, and relevant regulatory authority. This requirement may vary depending on the animal species used, the developmental stage (In this protocol, age of the zebrafish larvae and local legal requirements should particularly be concerned).
No tissue collection or dissection should be performed without the appropriate ethical approval, licence, or institutional permission where required.
Overview
This protocol describes the workflow for qRT-PCR-based gene expression analysis from animal tissues, including brief sample collection, RNA extraction, cDNA synthesis, qPCR setup, and ΔCt/ΔΔCt-based data analysis. The procedure was primarily used for zebrafish larval tissues, especially dissected eyes and brains. For other animal tissues, the same downstream workflow can be followed after appropriate tissue disruption and homogenisation.
Materials & Sample Collection
Sample Preparation:
Reagents: RNA Extraction Kit (QIAGEN, 74034), RT-PCR Kit (Thermo, Ref 4374966), qPCR Kit(Promega, USA, A6002)
Equipments: Centrifuge, Nanodrop, Thermocycler, qPCR Machine and consumables
Primers for RT-PCR and qPCR
RNA Extraction (QIAGEN 74034)
Prepare Buffer RLT Plus supplemented with β-mercaptoethanol by adding 10 µL β-mercaptoethanol per 1 mL Buffer RLT Plus. Keep all samples and RNA-containing solutions on ice where possible, and use RNase-free tubes, tips, and reagents throughout the procedure.
Dissect zebrafish larval brain tissue and immediately freeze the tissue on dry ice if extraction is not performed immediately.
Add 350 µL Buffer RLT Plus supplemented with β-mercaptoethanol to each sample.
Homogenise the tissue thoroughly using a 20-gauge needle and an RNase-free syringe. Pass the lysate through the needle multiple times until the tissue is fully disrupted and the lysate appears homogeneous.
If the homogenised lysate has been frozen, thaw the sample thoroughly using a water bath or metal bath for approximately 1 min 30 s before continuing with RNA extraction.
Centrifuge the lysate for 3 min at full speed to pellet debris.
Carefully transfer the cleared supernatant to a gDNA Eliminator spin column placed in a collection tube. Avoid disturbing or transferring any pellet material.
Centrifuge for 30 s at 12,000 rpm.
Keep the flow-through and discard the gDNA Eliminator spin column.
Add 350 µL 70% ethanol to the flow-through and mix well by pipetting. Do not centrifuge at this stage.
Transfer the ethanol-containing mixture to an RNeasy MinElute spin column placed in a collection tube.
Centrifuge for 15 s at 12,000 rpm and discard the flow-through.
Add 700 µL Buffer RW1 to the RNeasy MinElute column.
Centrifuge for 15 s at 12,000 rpm and discard the flow-through.
Add 500 µL Buffer RPE to the column.
Centrifuge for 15 s at 12,000 rpm and discard the flow-through.
Add 500 µL 80% ethanol to the column.
Centrifuge for 2 min at 12,000 rpm.
Discard the collection tube and place the RNeasy MinElute column into a new collection tube.
Centrifuge the column for 5–6 min at full speed to dry the membrane completely.
Place the RNeasy MinElute column into a new RNase-free 1.5 mL collection tube.
Add 14 µL RNase-free water directly to the centre of the membrane.
Centrifuge to elute RNA. Record the final RNA concentration and purity after elution.
Store purified RNA on ice for immediate reverse transcription, or at −80°C for longer-term storage.
cDNA Synthesis (Thermo, Ref 4374966)
Using the sample with lowest concentration to determine the total amount of RNA as input
V = 20ul
| Component | Volume per well | |
| 10x RT Buffer | 2 µL | |
| 25x dNTP Mix | 0.8 µL | |
| 10x RT Random Perimers | 2 µL | |
| inhibitor | 1 µL | |
| Transcriptase | 1 µL | |
| Nuclease-free water | n µL (making sure cDNA concentration is the same ) | |
| Diluted cDNA | n µL | |
| Total reaction volume | 20ul |
Following the system as kit suggests, and keeping the whole process on ICE !!!
After the reaction, DO NOT TEST CONCENTRATION OF RTPCR PRODUCT, STAIGHT TO QPCR
qPCR (Promega, USA, A6002)
qPCR plate setup design
Before setting up the qPCR reaction, plan a plate layout in Excel or the qPCR machine software. Each biological sample should be analysed with both the target gene primers and the housekeeping gene primers. Technical triplicates are normally used for each sample-primer combination.
Typical 96-well Plate Design:
| HSK | HSK | HSK | Gene 1 | Gene 1 | |
| WT1 | Replica 1 | Replica 2 | Replica 3 | ||
| WT2 | |||||
| WT3 | |||||
| Mut1 | |||||
| Mut2 | |||||
| Mut3 | |||||
| G | |||||
| H | NTC | NTC | NTC |
| Gene 1 | Gene 2 | Gene 2 | Gene 2 | Gene 3 | |
| WT1 | |||||
| WT2 | |||||
| WT3 | |||||
| Mut1 | |||||
| Mut2 | |||||
| Mut3 | |||||
| G | |||||
| H |
| Gene 3 | Gene 3 | |
| WT1 | ||
| WT2 | ||
| WT3 | ||
| Mut1 | ||
| Mut2 | ||
| Mut3 | ||
| G | ||
| H |
Prepare qPCR reaction mix
| Component | Volume per well | |
| 2x qPCR master mix | 10 µL | |
| Forward primer | 1 µL | |
| Reverse primer | 1 µL | |
| Nuclease-free water | 5.5µL | |
| Diluted cDNA | 2.5 µL | |
| Total reaction volume | 20 µL |
normally, I used to give make two mix for each well, (1+2+3) and (4+5).
similar volume should be taken, minimising the pipetting errors
Load and run the qPCR plate, follow the kit protocol
qPCR Data Analysis Flow
Export Ct values and perform quality control
Average technical replicates
Calculate ΔCt values
Calculate ΔΔCt and relative expression
Perform statistical analysis
Plot gene expression results