The overall goal of the following experiment is to conduct hybridization in C two for determining the localization and accumulation patterns of specific transcripts within mosquito tissues and embryos. This is achieved by first fixing the mosquito tissues or embryos so that the tissue ultra structure and mRNA targets will be preserved and available for hybridizing with antisense RNA probes. Next, the antisense RNA probes are hybridized to the target transcripts within the fixed tissues or embryos.
Then cholera metric antibody staining methods are performed in order to detect target mRNA localization and accumulation patterns. Results are obtained that show spatial patterns of RNA accumulation based on hybridization in C two and colormetric detection methods. This method can help answer key questions in the field of functional genomics, such as addressing when and where gene products accumulate in the mosquito.
This procedure also allows us to obtain spatial localization information to understand better how gene expression is regulated for biological processes such as reproduction development and mosquito pathogen interactions. To prepare for hybridizing digo oxygen and labeled RNA probes to mosquito tissues begin by dissecting the ovaries and salivary glands. Then fix the tissue and treat it with proteinase K if necessary.
After the tissues have been post fixed, equilibrate them in one milliliter of a one-to-one mixture of hybridization solution PBT or HIB PBT for 30 minutes with mutation, wait for the tissue to settle next decant as much HI PBT as possible. Then add one milliliter of HIB into each sample tube. Wrap the sample tube in sealed air protective packaging such as bubble wrap, and place it inside a hybridization bottle.
Seal the bottle with a hybridization bottle cap and incubate it at 55 degrees Celsius for 30 minutes with rotation. After removing the bottle from the incubator and allowing the tissue to settle carefully decant the pre hybridization buffer leaving 50 microliters of solution in the tube to prevent sample loss. Next, add 50 microliters of Prewarm high buffer to the same sample and place it in a heat block set to 55 degrees Celsius denature the previously prepared RNA probe at 85 degrees Celsius for five minutes.
Then immediately place it on ice for five minutes. Next, pipette the RNA probe into the sample tube. The RNA probe will float to the surface on the HI solution.
Mix by flicking the tube. Gently incubate the samples in a fixed micro fuge rack inside a hybridization oven at 55 degrees Celsius for 16 to 24 hours. Following RNAs, a treatment and antibody staining as outlined in the written protocol.
Mount the tissue samples by first using a 200 microliter pipette tip to transfer them into individual round bottom wells of a Pyrex spot plate. Remove as much of the PBT as possible and cover the sample with 70%glycerol in water. Allow the glycerol to permeate the sample at four degrees Celsius overnight.
To prepare microscope slides Begin by wiping the slide surface clean with Kim wipes. Next, apply two pieces of invisible tape parallel to each other so that they form a narrow channel using an artist's brush. Pick up samples one by one and place them along the length of the channel, positioning each sample with the same orientation with respect to anterior, posterior, and dorsal ventral alignments.
After positioning each sample, use a Kim wipe swab to absorb excess glycerol solution surrounding the tissues. Removal of the excess glycerol is important to prevent the formation of bubbles when sealing the mounted samples onto the slide under a cover slip, carefully place a cover slip over the channel containing the aligned samples Using transparent nail polish, affix the cover slip semi permanently onto the slide by dabbing the corners. Using a 200 microliter pipetter slowly dispense 70%glycerol into one end of the channel to fill the channel and area under the cover slip permanently.
Seal the mounted samples by applying nail polish along all four sides of the cover slip. Allow the nail polish to dry sufficiently before imaging the samples. After collecting embryos from a cage of 300 mated adult female mosquitoes and allowing them to develop to desired stages, transfer them from their collection container into a tylene mesh decoration Catch tube for eighties embryos.
Place the catch tube into a beaker that is filled with enough distilled water so that the volume of the catch tube is one half full. Use an artist's brush to dislodge the embryos from the tylene mesh and transfer them into the water filled catch tube. For an awfully embryos detach the tylene mesh from the collection container and fold the mesh carefully to form a funnel holding the mesh funnel with one hand.
Use the other hand to squirt water along the sides of the funnel to wash the embryos into the catch tube. This method can be applied to anomalies embryos because they lack the adhesive substance present on the surface of eighties embryos, which enables eggs to stick to over position substrates. Prepare 40 milliliters of decoration solution and pour the solution into a 100 millimeter Petri dish.
Add 50 milliliters of distilled water to a 100 milliliter beaker and set it aside. Next place the embryos into the Petri dish of dec decoration solution. Use a disposable polyethylene transfer pipette to wash the embryos while swirling the catch vial so that the embryos remain submerged and agitated in the decoration solution.
Decoration is one of the most sensitive steps of the protocol, and over extension of the sodium hypochlorite treatment of eighties embryos will prevent proper endo corion. Cracking a maximum of 35 seconds is required for eighties embryos while 75 seconds is sufficient for an awfully in QX embryos Under a dissecting microscope at low magnification verify decoration. Eighties embryos will lack the netting like exo corion and only the smooth polished surface of the black endo corion is present for anomaly's embryos.
The exo chorionic floats and ridge structures will be absent once the embryos are coated. Proceed to fix and disrupt the endo corion according to the procedure outlined in the written protocol. To peel off the disrupted endo Corion, place a piece of double-sided toupee tape in the center of a three centimeter Petri dish using a large bore 200 microliter pipette tip.
Transfer the embryos from methanol onto the double-sided tape. Allow one to two minutes for the embryos to settle and adhere to the tape surface decant excess methanol, and allow the surface to dry slightly for one to two minutes. Then add 95%ethanol into the Petri dish to further immobilize the embryos on the tape pipette 600 microliters of distilled water into the ethanol and swirl to mix.
Next, use a 27.5 needle attached to a one milliliter syringe barrel to peel off the cracked endo corion. After releasing the white embryo from the black endo corion remnants using a fine tip, artist's brush promptly transfer the embryos from the tape to the ethanol reservoir of the Petri dish. Then using a three millimeter or pipette tip, transfer the embryos into a 1.5 milliliter micro fuge tube containing 500 microliters of 100%Ethanol finally wash the embryos two to three times in 100%ethanol and store the embryos in punctilious ethanol at minus 20 degrees Celsius.
In this figure in C two hybridization of whole mount salivary glands of female eighties aegypti with mRNA probes that target amylase 1D seven S two and D seven L two show the accumulation of these transcripts in proximal lateral, distal lateral and distal lateral medial lobes respectively. In this experiment, whole Mount Cytes of three mosquito species were hybridized with mRNA probes that specifically target the respective autologous transcripts of Oscar. Here in C two hybridization of whole Mount embryos of Ananais Gambi eighties, a GTI and QX Qua Sitis was performed using antisense RNA probes targeting the respective mosquito Oscar autologous transcripts.
After watching this video, you should have a good understanding of how to perform a hybridization in C two experiment for determining the localization and accumulation of transcripts in specific mosquito tissues or embryos.
