Towards Quantitative In Situ Hybridization

Abstract

SUMMARYIn situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35 S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four-to fivefold in the same period, owing to a comparable decrease in the number of CPSexpressing cells in total intestinal tissue.

Stereological measurement of porto-central gradients in gene expression in mouse liver

“…16 The sections were simultaneously processed under well-defined conditions to ensure that the optical density (OD) of the autoradiography signals linearly reflected the cellular messenger RNA levels (mRNA). 16,17 The maximum OD did not exceed 0.7.…”

“…3) for the periportally expressed enzymes (A) PEPCK and (C) CPS, and for the pericentrally expressed enzymes (B) OAT and (D) GS. The OD values per zone are linearly related to the mRNA concentration 17 and can be used as a direct estimate of the relative level of gene expression. The affinity and cooperativity parameters that resulted when the gradients were fitted to a model for hepatic gene expression 2 are given in the table.…”

“…Tissue-mRNA content (silver staining) was recorded using white light. 17 The digital transmission images were converted to OD images by calculating the negative logarithm of the transmission image divided by an image of the light source: OD ϭ Ϫ 10 log(I/I 0 ) for each pixel. This conversion implicitly corrects background shading.…”

Dynamics of Hepatitis C Virus Replication in Human Liver

“…46 Various staining techniques have been used for quantification of cellular RNA and viral RNA in tissue specimens. 47,48 Haase and colleagues 49 reported the measurement of HIV genomes in lymphoid tissues by counting silver grains after hybridizing HIV genomes with 35 S-labeled anti-sense riboprobes and autoradiographic exposure. Chemiluminescent in situ hybridization method was demonstrated measuring cytomegalovirus and human papillomavirus genomes in tissue specimens.…”

Proteinuria and the expression of the podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effects of angiotensin converting enzyme inhibition

“…Quantitative in situ hybridization which permits the assessment of gene expression equivalent to northern blot analysis [11], was used to determine the magnitude of gene expression, as previously described for the analysis of gene expression in the kidney [12]. In brief, a 293 base pair cDNA coding for human nephrin was cloned into pGEM-T (Promega, Madison, Wis., USA), linearized with Not I and an antisense riboprobe was produced using T7 RNA polymerase.…”