Dear Rita

Do you want to extract infectious virus or viral nucleic acid, I didn't get it from your question. For extraction of infectious virus, you have to homogenized the mosquito pool in liquid nitrogen or chilled MEM in ice. Centrifuge it and take the supernatant and passed it through 0.22 micron filter and infect on C6/36 and BHK cell. For nucleic acid extraction fallow this links

http://www.ncbi.nlm.nih.gov/pubmed/23390520

http://www.ncbi.nlm.nih.gov/pubmed/23326348

@ Pradip Fulmali: Sorry the question I posted was not clear enough. I have experience working with Anopheles, baseline suseptibility against pesticide.. resistant gene expression studies.. I dont have prior knowledge and experience on extraction of infected culicine mosquitoes; or rather extraction of virus from infected Culicine mosquitoes...

Is there any other method of not using dry ice? Im afraid, dry ice of scarce in our region. Can you please elaborate on the use of MEM?

thank you again

regards

Rita

If you want to look for genetic markers, or material for sequencing, you can process the homogenized mosquitoes immediately with with reverse transcriptase, and extract the corresponding DNA later.

Thank you Gunnar

Hi Rita, There are protocols for the extraction of flavivirus in mosquitoes, in terms of the field is good to have liquid nitrogen tank to store the material and then manipulate in cold table, and then make the pools, we extract these cold pools using Qiagen RNeasy kit, when the RNA has taken two aliquots, one is saved and the other is used for RT-PCR molecular detection's another way you could do is soak the mosquito pool with minimum essential medium homogeizar and then centrifuge and aliquot be saved and one for RNA extraction. I am attaching a pdf that talks about protocols for this type of research, however, they are applicable to dengue. This just the beginning of the chapter, but the title can lead to download it to Springer.

Saludes from Colombia, I'm starting a Dengue hard work!

We extract RNA from mosquito pools using the PureLink Micro-to-Midi Total RNA Purification System (Invitrogen) or Viral RNA kit (QIagen) or if you have available you can also use a automated extraction system, as QIacube, EasyMag or equivalent (usually with a kit specific for viral nucleic acids you will achieve better results than using Total RNA extraction, but both work). RNA extraction should be performed according to the manufacturer’s recommendation for frozen or fresh fibrous animal tissue. We macerate the mosquitoes in cold environment by adding liquid nitrogen and using RNase-free mortar and pestle for lysis and then adding 1 ml PBS plus fetal calf serum. Half of the homogenate from each mosquito pool is stored at -80ºC for later viral isolation (if necessary). And the other half proceed for extraction. Using dry ice is important to achieve a better homogenate and to prevent RNA degradation. However, if you really do not have that possibility I would choose to frozen the mosquitoes at -80ºC and then quickly macerate then with cold mortar and pestle (at -20ºC) and placed in ice, and then proceed directly to extraction. Good luck!

We ground mosquito by vortexing the sample with round copper beads (bullets for air pistol) and pbs. Then we extract RNA with Trizol. A table with methods utilized by different groups is present in mat and met of

http://www.ncbi.nlm.nih.gov/pubmed/22377581

Kind regards

Mattia

Thank you so much.

If you are interested in looking for arbo- viral infection status in mosquitoes you may use the traditional methodology - TRI reagent RT (Molecular Research) for extraction of total RNA from mosquitoes. After that you could carryout RT-PCR with specific DNA primers for Alphaviruses/Flaviviruses. I am attaching one of my papers on detection of infection of CHIKV in Aedes albopictus. Also we use the same extraction methodology for DENV. The only difference being usage of with DENV specific DNA primers for RT-PCR. It also works.

Thank you Sir

The mosquito pools are to be homogenized in chilled MEM in ice. The supernatant collected to be filtered and can be cultured in C6/36 cell line. The viruses from the cultures can be used for any analysis. lalitha kabilan

I removed wings and legs of female mosquitoes and thorax and heads in each pool were macerated in 300 ul of DMEM culture medium with antibiotics (penicillin-streptomycin) and fetal bovine serum. Samples were centrifuged and supernatants used to purify RNA using the QIAmp mini Viral RNA kit and stored at -80 ºC until further use.  

Jaime Castellanos, how many mosquitoes do you use in each pool?

Dear N Pradeep Kumar,

Sorry for disturbing and I am Lee. I would like to know that you mentioned using TRI reagent for RNA extraction is it TRIZOL reagent that you mentioned above?  and may I know when using the TRIZOL reagent to conduct the RNA extraction from the adult mosquito, the extracted RNA is secondary or linear structure? 

Hi Florencia,

We use normally 20 females (without wings and legs) each eppendorf tube.

I like to use a teflon pestle for eppendorf. This supernatant could be used in both isolement on C6/36 cells and to extract RNA to RT-PCR. (Perez-Castro et al, Mem Inst Oswaldo Cruz, 2016;111: 233-240)

Dear Jaime Castellanos,

why do you take away wings and legs? Isn't there probability of dissemination of the virus in the legs? 

Hai,

Whole mosquitoes is fine. If you remove one or two legs of the specimen for extraction of DNA you could confirm the species of mosquito by DNA Barcoding methodology.

Dear Jaime Castellanos,

Can I use Eagles medium, and is it essential to use antibiotics and FBS to homogenize the mosquitoes for RNA extraction

Dear all,

I am trying to detect the presence of dengue virus in field caught Aedes aegypti and Aedes albopictus using the Promega total RNA extraction followed by RT PCR with DENV specific primers (Lanciotti et al). however i am continuosly getting negative results. i useed 5-20 mosqutioes per pool - head and throxes only. i am afraid when i separate the head and throxes of a pool of 20 mosquitoes which takes some time is there a possibility of RNA degration of the separated ones? when the whole mosquito is taken it tends to block the column when extracting. Could someone tell me what the precautions i can follow?

thanks a lot

Hi.. my study is on the surveillance section (WNV detection on mosquito). Is it enough if I put only PBS buffer on pooled mosquito to homogenize and proceed to RNA extraction using Trizol reagent? I will use pellet pestle cordless motor for homogenization

After RNA Extraction, I will carry out nested PCR.

Thank you

Hi Natasha Jafar Ali ,

I also use only PBS (150ml) to homogenize mosquitoes, then I proceed with Trizol for RNA extraction.

thank you Melisa Berenice Bonica for the response... Did you refer to any journal on this method? May I know how many mosquitoes did you pool for each sample? Did you use pellet pestle as well?

Thank you

Natasha Jafar Ali , mosquitoes pools vary depending on the mosquito species. For Ae. aegypti or albopictus we don't exceed 10 mosquitoes per pool. I suppose you are working with Culex, I would use 5/6 mosquitoes as max. I don't refer to a journal, only suggestions from other people. And yes, I use pellet pestle to homogenize with PBS.

Are you sure your mosquitoes are infected? Maybe you don't have positive results because they aren't infected.

Yes, I am working with Culex so far. The mosquitoes are not infected directly and I collected from selected animal habitat and will proceed to WNV detection after species identification. Pertaining to your answer, does that mean you add PBS 150 ml for one pooled sample (5/6 mosquitoes)? how about the homogenization procedure? how long you homogenize with pellet pestle and did you centrifuge it at 4-degree Celcius? Sorry for too many questions. This study is something new in my place.

Thank you Melisa Berenice Bonica for your help.

Natasha Jafar Ali I sent you a private message.

Natasha Jafar Ali I sent you a private message.

Thank you Melisa Berenice Bonica for the message.