I am a master student under virology program. Currently, I am working on determination of virus among mosquitoes population . Based on previous study, most of them used bead-based homogenization to homogenize mosquitoes before extraction steps. However, i would like to inquire, is there any published study on using rotor stator homogenization such as mechanical homogenizer without using beads to homogenize mosquitoes. For now, there are no other equipment to homogenize mosquitoes except mechanical homogenizer and beads are quite expensive to purchase. With that concern, I would like to modify the homogenization technique correspond to available equipment in my laboratory. Your response are kindly appreciated.
Thanks for helping me.
Hi Natasha,
I have used a bead beater when I am extracting large numbers of samples e.g. in a 96 well plate on a Qiagen bead beater. If you have no bead beater, or small numbers of samples then you can extract individually. When I perform DNA extraction from small numbers of samples I use a plastic pestle in an Eppendorf tube (e.g. https://www.fishersci.co.uk/shop/products/fisherbrand-rnase-free-disposable-pellet-pestles-2/13236679). You can grind these manually or get a battery powered grinder to attach the pestle to.
Pestles can be reused if they are cleaned, sterilized and autoclaved between uses.
Hello Natasha,
During my master in Virology, for all the mosquito experiment we performed, i have used a hand held mechanical triturator. Its a simple device with a motor and a detachable plastic triturator which can be attached to the end and is battery operated.
The homogenization of mosquitos was done in Eppendorf tubes and furhter experimentation using the obtained tissue homogenate and the supernatant fluids using immunofluoresence give excellent results.
A mechanical tissue homogenizer would also suffice your current need for mosquito homogenization.
All the Best!
Regards,
Akshay
Hello Natasha,
You can use a Dounce homogenizer to disrupt the mosquito cells in the presence of low concentration of detergent (Triton X-100 or NP-40). We use this method and it usually gives good yield of DNA.
Good luck!
Jagaree
Hello Natasha,
Your can use simple mechanical tissue homogenizer it will solve your purpose. I had extracted digestive enzymes from the gut of mosquito larvae and got my results.
Good luck.
Sure Natasha, use a mechanical homogeneizer by hand or electrical, depending of the number of mosquitoes samples yo have, you can use one or the other. Also you can use a microplate to put all your mosquitoes there and homogeneize one by one.
Hello..
Thank you researchers for sharing your wisdom with me. I really appreciate your guidance and time. I will try the above method and find out the best procedure for my study.
Thank you
Do I need to centrifuge after using hand-held homogenizer? is there any paper that conducting this homogenization method which is mechanical homogenizer? I would like to know the duration of the process and other important details on this method.
Thank you for your help
Hi there , Yes I recommend to centrifuged the sample for 10 min at 10,000 rpm (with a temperature of 4C). Usually, we macerated organisms until no body parts are recognized (more or less one minute).
Hi, thank you Miguel Moreno-García for the response. I appreciate it. However, if I centrifuge it longer or shorter than the one that is recommended, will that affect the sample like shorter rpm will degrade the RNA, for example?
Thank you
Is anyone familiar with BA-1 diluent as referred to by Lanciotti and others for mosquito homogenization? I'm looking for a protocol to make it. Thanks.
Hello sir Craig S Wilding, have you ever use pellet pestle cordless motor to homogenize the mosquito? If yes, may I know what reagent did you put before homogenize the pooled mosquito? Thank you
Hi.. Sir Akshay Deshpande .. pertaining to your answer, may I know the solution/reagent that you added before homogenize the pooled mosquito? I will extract the RNA with trizol..
thank you
Hi Natasha,
I used to resuspend the tissue homogenate in PBS. It sufficed my purpose since I was aiming to isolate viral proteins and perform immunofluoresence form the sample. in case of RNA isolation, i would recommend you perform the experiment using RNA isolation (tissue lysis) buffers provided with RNA extraction kits. You can us the trizol in the second step of the extraction procedure.
Good Luck!]
Akshay
Thank you Sir Akshay Deshpande for your answer.. My study is on the surveillance section (WNV detection on mosquito). Is it enough if I put only PBS buffer on pooled mosquitoes (1-10 mosqui per sample) to homogenize and proceed to RNA extraction using Trizol reagent? I am using pellet pestle cordless motor for homogenization After RNA Extraction, I will carry out nested PCR. Thank you
Hello again,
Yes. I think the extraction part can be done in PBS which should not harm any nucleic acids. Just make sure the water you are using for the preparation of PBS is nuclease free and also make sure that the PBS is sterile (mostly filter-sterilized through 0.22micron filter), since you do not want any interference for your next step of extraction.
You can also perform a centrifugation step at least two times to completely remove the debris from the homogenization of mosquito.
Good Luck!
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