Fluorescent In Situ Transcription in Cells and Tissues

互联网2013-12-31

915

Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This method assesses cellular mRNA transcripts in tissue sections and cell cultures using unique short anti?sense primers directed against sequences in particular protein(s). The unlabeled synthetic cDNA oligonucleotide primers are extended complementary to a sense mRNA transcript using reverse transcriptase and labeled through incorporation of a fluorescent?labeled dUTP nucleotide base. The new cDNA will be synthesized upstream from the point of primer hybridization, and has a specific activity of fluorescent labeling dependent upon the length of the template mRNA from the primer location to the 5'?terminus. This procedure provides rapid detection of low abundance mRNA messages that can be related to other cellular protein components, labeled experimentally with alternative fluorochromes.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Basic Protocol 1: Fluorescent In Situ Transcription
Reagents and Solutions
Commentary
Figures

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Fluorescent In Situ Transcription

Materials

Slide‐mounted tissue sections or cultured cells
4% (w/v) paraformaldehyde (Sigma) in sterile PBS (see recipe ), made fresh
PBS ( appendix 2A )
20× SSC (Sigma or appendix 2A )
0.001% (v/v) digitonin (Sigma) in 2× SSC
Blocking buffer (see recipe )
Prehybridization buffer: 50% formamide (Sigma) in 2× SSC
Hybridization buffer: 50% formamide in 1× SSC
Experimental primers: unique DNA oligonucleotide primers (custom synthesis, 36‐ to 39‐mer; )
Control primers: e.g., missense primers (custom synthesis, 36‐ to 39‐mer) and oligo(dT)₃₆ primer (USB); see
RT buffer (see recipe )
PAP Pen (hydrophobic slide marker; Research Products International)
Moist chamber: sealable plastic container (e.g., Tupperware) containing a water‐saturated foam pad insert
Fluorescence microscope with appropriate dichroic filters to detect chosen fluorochrome(s)
Camera or electronic image‐analysis system to record data

NOTE: Use DEPC‐treated double‐distilled water ( appendix 2A ) for all reagents in pretreatment and hybridization steps. All materials should be autoclaved and RNase free.NOTE: See for a detailed discussion of necessary control experiments.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 5.13.1 Flow chart of the FIST method.

View Image

Figure 5.13.2 Experimental primer specificity for determination of D1_A dopamine receptor expression using a mutant mouse deficient in the receptor subtype (Drago et al., ). The specific primer generated was complementary to 36 oligonucleotide bases at the carboxyl end of the receptor mRNA sequence (Monsma et al., ). (A ) Transcript staining is evident as bright fluorescent neurons (arrows) of medium diameter in this 10‐µm‐thick frozen/fixed tissue section of the wild‐type (WT) mouse striatum. (B ) Fluorescence is diminished in the mutant knockout (KO) mouse. Tissue processing, visualization, photomicrography, and dark‐room enlargements were made concurrently for the WT and KO sections, using identical procedures and settings.

View Image

Videos

Literature Cited

Literature Cited
	Ariano, M.A., Larson, E.R., Noblett, K.L., Sibley, D.R., and Levine, M.S. 1997a. Coexpression of striatal dopamine receptor subtypes and excitatory amino acid subunits. Synapse 26:400‐414.
	Ariano, M.A., Wang, J., Noblett, K.L., Larson, E.R., and Sibley, D.R. 1997b. Cellular distribution of the rat D4 dopamine receptor protein in the CNS using anti‐receptor antisera. Brain Res. 752:26‐34.
	Drago, J., Gerfen, C.R., Lachowicz, J.E., Steiner, H., Hollon, T.R., Love, P.E., Ooi, G.T., Grinberg, A., Lee, E.J., Huang, S.P., Bartlett, P.P., Jose, P.A., Sibley, D.R., and Westphal, H. 1994. Altered striatal function in a mutant mouse lacking D1A dopamine receptors. Proc. Natl. Acad. Sci. U.S.A. 91:12564‐12568.
	Monsma, F.J. Jr., Mahan, L.C., McVittie, L.D., Gerfen, C.R., and Sibley, D.R. 1990. Molecular cloning and expression of a D1 dopamine receptor linked to adenylate cyclase activity. Proc. Natl. Acad. Sci. U.S.A. 87:6723‐6727.
	Noblett, K.L. and Ariano, M.A. 1996. Co‐expression of receptor mRNA and protein: Striatal dopamine and excitatory amino acid subtypes. J. Neurosci. Methods 66:61‐66.
	Noblett, K.L. and Ariano, M.A. 1998. Detection of receptor mRNA using fluorescent in situ transcription (FIST). In Receptor Localization: Laboratory Methods and Procedures (M.A. Ariano, ed.) pp. 182‐196. John Wiley & Sons, New York.
	Tecotte, L.H., Barchas, J.D., and Eberwine, J.H. 1988. In situ transcription: Specific synthesis of complementary DNA in fixed tissue sections. Science 240:1661‐1664.
Key Reference
	Tecotte et al., See above.
	This article describes the initial development of in situ transcription using radiolabeled probes. It demonstrates the feasibility and ease of using this method versus standard in situ hybridization to detect transcripts.