title: Comparison of RNA isolation methods from insect larvae.
abstract: 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.
authors.affiliation: Department of Zoology and Entomology, Rhodes University, P.O. Box 94, Grahamstown 6139, South Africa jarydridgeway@gmail.com.
authors.forename: J A
authors.initials: JA
authors.lastname: Ridgeway
authors.affiliation: Department of Zoology and Entomology, Rhodes University, P.O. Box 94, Grahamstown 6139, South Africa.
authors.forename: A E
authors.initials: AE
authors.lastname: Timm
mesh_terms: D000818:Animals; D001517:Coleoptera / Q000235:genetics* / Q000254:growth & development; D007814:Larva / Q000235:genetics; D009036:Moths / Q000235:genetics* / Q000254:growth & development; D016133:Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods*; D013712:Tenebrio / Q000235:genetics / Q000254:growth & development
keywords: CTAB; RNA extraction; RNeasy Mini Kit; SV Total RNA isolation system; TRIzol reagent