Yonsei Med J.  2015 Mar;56(2):497-502. 10.3349/ymj.2015.56.2.497.

A Melting Method for RNA Extraction from the Mucosal Membrane of the Mouse Middle Ear

• Seo YJ
• Kim SH
• Moon IS
• Choi JY

• KMID: 2070030
• DOI: http://doi.org/10.3349/ymj.2015.56.2.497

Abstract

PURPOSE
There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use.
MATERIALS AND METHODS
Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach.
RESULTS
Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM.
CONCLUSION
This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.

Keyword

Melting; RNA extraction; middle ear mucosa; mouse

MeSH Terms

Animals
Ear, Middle/*metabolism/pathology
Mice
Microscopy, Electron, Scanning
Mucin 5AC/genetics/*metabolism
RNA, Messenger/*genetics/metabolism
Reverse Transcriptase Polymerase Chain Reaction/*methods
Spectrophotometry
Mucin 5AC
RNA, Messenger

Figure

• 

  Fig. 1 Lateral postauricular approach to the mouse middle ear (bulla). (A) In each animal, a postauricular incision (black line marker) was made and extended ventrally to the rostral neck skin. The subcutaneous connective tissues were separated to expose the deeper structures. (B) The incision was deepened to the underlying temporalis and cervical muscles. Subsequently, the submaxillary gland was carefully dissected out and retracted, working across the sternocleidomastoid muscle (black arrow) and the digastric muscles (arrowhead). The bulla (star) is the posterior insertion of the digastric muscle into the jugular process of the occipital bone. (C) Access to the tympanic bulla was achieved by means of a blunt dissection slightly lateral to the insertion of the digastric muscle. The figure shows the bulla (black arrow) connected to the EAC (green arrow). (D) The EAC was resected with the tympanic membrane using a probe that was inserted into the EAC and bulged toward the dissection. The previously exposed bulla (green arrow) is inferior to the resected tympanic membrane, showing the middle ear cavity inside (white arrow). (E) Looking through the hole of the bulla upside the mouse, the half-exposed clean bulla (black lines) was resected with sharp micro-scissors, leaving no fragments. (F) After the resection, the medial wall of the bulla, including the promontory (black arrow), remained. EAC, external auditory canal.

• 

  Fig. 2 Light micrographs of the middle ear mucosa in the whole bulla. (A) Inside the bulla bone, ciliated mucosal membranes were evenly distributed. A lateral approach exposed the half lateral bulla (black box) (×10). (B) The resected lateral bulla had securely attached mucosal membranes (×20, ×400).

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  Fig. 3 Scanning electron micrographs of the middle ear mucosa in the resected bulla. (A) Before melting, the picture shows ciliated mucosal cells regularly distributed on the surface of bony bulla (×1000). (B) A bundle of cilia on the hexagonal cell (×10000). (C and D) After melting the membrane into the TRIzol solution for 10 minutes, it showed uneven surfaces and no ciliated cells (×1000, ×10000). There were very few mucosal cells on the surface, and it melted almost all mucosal cells of the bulla, not exposing bony surfaces. (E and F) After stripping, the bony surface was exposed with some bony scratching (×1000, ×10000).

• 

  Fig. 4 Expression of mucosal marker (MUC5AC) and bony marker (osteocalcin) in mouse middle ear epithelium by RT-PCR; 518-bp and 371-bp fragments amplified on the RT-PCR probed by MUC5AC and total osteocalcin primers. MUC5AC was detected in the cells within 1 hour, while MUC5AC and osteocalcin were simultaneously detected over a period of 1 hour. RT-PCR, reverse transcription-polymerase chain reaction; M, MUC5AC primer in the melting method; O, osteocalcin primer in the melting method; Ms, MUC5AC primer in the stripping method; Os, osteocalcin primer in the stripping method.

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