( ...)Detection of mRNAs on Cryosections of the,,,Cardiovascular System Using DIG-Labeled RNA Probes

The following protocol was optimized from a protocol using 35S-labeled RNA probes [1, 2]. In research studies, it enables detection of the expression of rare mRNAs in the cardiovascular system (e.g., of the proinflammatory cytokine granulocyte macrophage colony stimulating factor [GM-CSF] in normal human coronary arteries, and of interleukin 6 [IL6] and glycoprotein gp130 in failing human hearts [3]). The protocol can be combined with immunohistochemistry [4, 5], and can also be used for paraffin- and methacrylate-embedded sections.

Materials and Methods

Preparation of DIG-labeled RNA probes

The desired cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR), and the cDNA was cloned into an in vitro transcription vector. DIG-labeled RNA probes were transcribed in vitro from the plasmid according to the instructions of the DIG RNA Labeling Kit. Best results will be obtained with digoxigenin-labeled RNA probes of < 600 bp. For larger probes, adjusting the proteinase K digest is preferred to probe denaturation. The amount of DIG-labeling was determined by dot blot. It is important to determine the exact concentration of the RNA probes to avoid background after hybridization.

Tissue preparation

Tissue was frozen as soon as possible after excision to prevent degradation of mRNA. The tissue was cut to the appropriate size, and as much of the fatty tissue as possible was removed. The tissue was then immersed in a cryoprotective medium and frozen on cork disks in nitrogen- cooled 2-methylbutan. The tissue was stored at 80 C or in liquid nitrogen.

Preparation of cryosecti
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