Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The overall goal of this procedure is to collect hearts by rapid manual dissection from live zebrafish embryos to obtain cardiac specific Mr.NA.This is accomplished by first pipetting, many anesthetized embryos up and down through a narrow pipette tip to simultaneously release the hearts from the embryonic bodies. Next, the dissected hearts are separated from embryonic debris in two rapid filtration steps. Then the fluorescently labeled hearts are manually sorted from remaining debris.

Finally, cardiac specific RNA is extracted from collected hearts. Ultimately, the heart dissection protocol is used to collect sufficient amounts of embryonic hearts to perform transcriptome analysis. This method will help to ask key question in the silverfish heart developmental field, such as which transcript are expressed within the heart at different developmental stages, and also compare wild and mutant heart expressions To obtain zebrafish embryos for heart extraction, cross adult transgenic fish expressing a fluorescent protein in the heart, such as transgenic mile seven EGFP under the stereo microscope.

Monitor the development of the embryos. Verify that the hearts are indeed fluorescently labeled and manually dec coate the embryos when the embryos have reached the desired stage. Transfer around 100 embryos into a 1.5 milliliter fuge tube and add point 16 milligrams per milliliter of trica in E three medium to anesthetize them.

Put the tube on ice. Remove the trica solution and use one milliliter of ice cold L 15 10%FBS medium to wash the embryos, keeping them on ice. After removing this wash, add one milliliter of L 15 FBS and use a round gel loading tip to pipette the embryos up and down five to eight times in order to completely disrupt the yolks, pipette the embryos dropwise onto the surface of the solution so that the drop will burst and the embryos are gently disrupted.

For this first batch of dissected embryos under a stereo microscope, assess their integrity and the proportion of dissected hearts and adjust the manner of pipetting accordingly. For subsequent batches, place a 100 micrometer filter onto a 50 milliliter centrifuge tube and apply the embryonic sample. Then use one milliliter of L 15 FBS to rin the fuge tube and apply the solution to the filter to flush out the hearts that remained in the filter.

Use L 15 FBS to wash it twice. Next, place a 30 micrometer filter onto a 15 milliliter centrifugation tube and apply the flow through containing the hearts. Use L 15 FBS to rinse this filter, which retains the hearts and wash off smaller debris.

Then turn the 30 micrometer filter upside down over an agros coated Petri dish, and apply three one milliliter washes of L 15 FBS medium to flush the hearts out of the filter Under a fluorescent microscope, use a pair of forceps to manually separate GFP positive hearts from non fluorescent tissues, concentrating them into the center of the dish. The physiology of the hearts appears normal as they are still beating after the dissection.Procedure. To isolate mRNA from the hearts, collect them in the smallest possible volume of medium and pipette them into a 1.5 milliliter tube on ice containing 0.75 milliliters of RNA.

Later under the fluorescent microscope. View the pipette tip to verify that no hearts remain stuck to the pipette tip. After pulling the hearts from several rounds of isolation into the tube of RNA later, according to the text protocol, centrifuge's samples at 15, 700 Gs and four degrees Celsius for 20 minutes.

Next, under a fluorescent microscope, carefully collect as much sate as possible and transfer into a 1%agros coated Petri dish containing three milliliters of L 15 10%FBS medium. Next, under a chemical hood, add 0.5 milliliters of triol to the tube containing the sedimented hearts and keep on ice Under the fluorescent microscope, transfer the hearts that remained in the supernatant into a fresh 1%agros coated dish with L 15 10%FBS to dilute out the RNA later, which is very viscous. Then collect the hearts in a small volume and transfer them into the tube of sedimented hearts in triol to disrupt the hearts, vortex the tube and incubate for five minutes at room temperature.

Then under a chemical hood, add 100 microliters of chloroform mix thoroughly and transfer the solution into a 1.5 milliliter PrepU, PLG tube Incubate for three minutes at room temperature, centrifuge a sample at 15, 700 Gs and four degrees Celsius for 15 minutes, and transfer the aqueous phase into a new 1.5 milliliter tube. Then add five to 10 micrograms of glycogen and 250 microliters of pre cooled isopropanol. Mix well and incubate overnight at minus 20 degrees Celsius the following day.

Centrifuge the samples at 15, 700 Gs and four degrees Celsius for 30 minutes, and discard the snat. Apply one milliliter of 75%ethanol to wash the palette centrifuge and discard the snat. Allow the remaining ethanol to evaporate from the pellet at room temperature until the pellet becomes white.

Add 20 microliters of RNAs free, double distilled water to the pellet and incubate at 55 degrees for 10 to 15 minutes to dissolve the RNA. Then place the tubes on ice. Use a spectrophotometer to determine the RNA concentration and purity and run a sample on a 1%agros gel to assess the integrity of the RNAA comparison of the heart samples prior to sorting from embryonic debris at 56 hours post fertilization versus 36 hours post fertilization is shown here.

Embryos at 56 hours post fertilization yielded more embryonic debris than at 36 hours post fertilization following filtration to determine the purity of the 56 HPF heart sample, the expression levels of cardiac versus extra cardiac transcripts were compared by R-T-Q-P-C-R. This gel shows RNA extracted from the isolated hearts and from the disrupted embryos without hearts. Absorbance measurements revealed that 300 hearts yielded around 660 nanogram RNA.

That is 33 nanogram per microliter in a final volume of 20 microliter. Relative gene expression levels were calculated by R-T-Q-P-C-R experiments for the markers shown here. As expected mile seven was highly enriched in the heart sample as compared to the embryo without heart sample as seen here.

The endothelial cell marker, KDRL was moderately enriched in the heart. The erythrocyte marker hemoglobin beta embryonic 1.1 was overrepresented in the heart sample, even though the hearts were still beating after dissection, which should expel red blood cells from the heart. In contrast levels of the lens marker alpha A crystalline and the CNS marker, GFAP were considerably reduced in the heart sample.

These results show that the heart dissection protocol yields high quality cardiac specific RNA from whole zebrafish embryos. For this experiment, it's important to monitor the sample in order to avoid losing hearts during the dissection procedure.