Dear Sir. Concerning your issue about the RNA extraction from mosquitoe larval sample .

Isolating RNA from insects is becoming increasingly important in molecular entomology. Four methods including three commercial kits RNeasy Mini Kit (Qiagen), SV Total RNA isolation system (Promega), TRIzol reagent (Invitrogen), and a cetyl trimethylammonium bromide (CTAB)-based method were compared regarding their ability to isolate RNA from whole-body larvae of Thaumatotibia leucotreta (Meyrick), Thanatophilus micans (F.), Plutella xylostella (L.), and Tenebrio molitor (L.). A difference was observed among the four methods regarding RNA quality but not quantity. However, RNA quality and quantity obtained was not dependent on the insect species. The CTAB-based method produced low-quality RNA and the Trizol reagent produced partially degraded RNA, whereas the RNeasy Mini Kit and SV Total RNA isolation system produced RNA of consistently high quality. However, after reverse transcription to cDNA, RNA produced using all four extraction methods could be used to successfully amplify a 708 bp fragment of the cytochrome oxidase I gene. Of the four methods, the SV Total RNA isolation system showed the least amount of DNA contamination with the highest RNA integrity number and is thus recommended for stringent applications where high-quality RNA is required. This is the first comparison of RNA isolation methods among different insect species and the first to compare RNA isolation methods in insects in the last 20 years. I think the following below links may help you in your analysis:

https://www.ncbi.nlm.nih.gov/pubmed/25527580

https://www.labome.com/method/RNA-Extraction.html

http://www.ijstr.org/final-print/dec2016/Isolation-Of-Total-Rna-Silkworm-bombyx-Mori-L-Larvae-lepidopterabombycidae-C301-On-Their-Several-Body-Tissues.pdf

Thanks

Hello,

I always use fresh samples for RNA extraction. Also, the second issue could be due to not adding the right amount of trizol in comparison to the amount of tissue. So, check whether you are adding it right. May be, this is why the third issue is arising. Third issue can also be due to the negligence during pipetting out the upper aqueous phase.

Thanks to Isam Eldin Hussein Elgailani and Vaishali Rani for your valuable comments.

Vaishali I am using the fresh sample only, but will the samples containing the water residue due to experimental setup will not hamper the RNA during homogenization. Can you just brief me with sample processing for homogenization if you are working in same area of RNA isolation from mosquito larval samples

Prabhakar, I am sorry I cannot help with you that because I have never extracted RNA from the mosquito larval samples. But I don't think there is any need to process your samples because Trizol reagent already contains Guanidinium thiocyanate which is already a protein dentaurant.

Hope, it helps you.