title: Evaluation of RNA quality in fixed and unembedded mouse embryos by different methods.
abstract: Many miRNAs are highly expressed in spatiotemporal and precise tissue-specific patterns in development. Thus it is necessary to examine their expression pattern in mouse embryos. However, embryos from one pregnant mouse are more than enough for expression analysis such as RT-qPCR, which results in reluctant disposal of remaining embryos. Due to the limitation of short sampling time, it is vitally important to quickly preserve samples to ensure the RNA quality. Thus, it is necessary to develop appropriate methods to fix samples in advance. In this study, two fixatives [methanol/DMSO (4:1) and paraformaldehyde] were applied for embryo (12.5 dpc) fixation and two preservatives (methanol and 30% sucrose) were used for fixed embryo preservation. After storage for one month, the skin, skeletal muscle and brain tissues were dissected from the fixed and unembedded embryos. Total RNAs were extracted by TRIzol® reagent and measured by a spectrophotometer, then were subjected to amplify Actb, Hprt, Gapdh, Rnu6, Snord68 and miR-206-3p by RT-qPCR. Embryos fixed in methanol/DMSO and preserved in 100% methanol at -20°C were able to yield at least 349 bp amplifiable RNA. Although paraformaldehyde fixation and 30% sucrose preservation method only yielded amplicons less than 156 bp, it showed a remarkable ability in preserving small RNAs. Snord68 was expressed stably across skin, skeletal muscle and brain tissues like Rnu6, making its possibility as an internal control for qPCR data normalization. Using Snord68 and/or Rnu6 as internal control, we found that the miR-206-3p expression level in skin was about one quarter of its highest level in the skeletal muscle. Therefore, the techniques in this study would be useful for us to reasonably utilize and preserve precious samples.
authors.affiliation: Department of Clinical Laboratory and Hematology, School of Medical Science and Laboratory Medicine of Jiangsu University, Zhenjiang, Jiangsu 212013, China; Department of Clinical Laboratory, Nanjing Children's Hospital, Nanjing Medical University, Nanjing, Jiangsu 210008, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Mu
authors.forename: Hong
authors.initials: H
authors.lastname: Zhou
authors.forename: Wenyan
authors.initials: W
authors.lastname: Li
authors.forename: Lichao
authors.initials: L
authors.lastname: Hu
authors.forename: Yiting
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D004121:Dimethyl Sulfoxide; D004622:Embryo, Mammalian / Q000737:chemistry; D005404:Fixatives; D005557:Formaldehyde; D000432:Methanol; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000032:analysis*; D011108:Polymers; D020871:RNA Stability; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013395:Sucrose; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D005404:Fixatives; D035683:MicroRNAs; D011108:Polymers; D005557:Formaldehyde; D013395:Sucrose; C003043:paraform; D000432:Methanol; D004121:Dimethyl Sulfoxide
keywords: Embryo; Fixation; MicroRNAs; Preservation; RNA quality; Real-time polymerase chain reaction