[Abstract]  [Full Text]  [PDF]

Jack M. Gallup^1*, Kenji Kawashima², Ginger Lucero³ and Mark R. Ackermann¹

¹ Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University. Ames, Iowa 50011-1250. USA.
² Environmental Hygiene Section, Shichinohe Research Unit, National Institute of Animal Health. 31 Uminai, Shichinohe, Aomori 039-2586. Japan.
³ Invitrogen R&D. Carlsbad, CA 92008. USA.

* To whom correspondence should be addressed: Jack M. Gallup, Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University. Ames, Iowa 50011-1250. USA. Email: eag@iastate.edu

Biol. Proced. Online 2005;7:70-92. doi:10.1251/bpo107
Submitted: March 21, 2005; Accepted: May 12, 2005; Published: May 30, 2005.

Indexing terms: Immunohistochemistry; Sheep; Gene Expression; Polymerase Chain Reaction.

We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead.