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Anal Biochem. 2014 Feb 15;447:126-32. doi: 10.1016/j.ab.2013.11.015. Epub 2013 Nov 20.

Laser capture microdissection after γ-glutamyl transferase histochemistry: an optimization for gene expression analysis.

Author information

1
Instituto Nacional de Medicina Genómica, México, Distrito Federal, Mexico; Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del I.P.N, Distrito Federal, Mexico.
2
Instituto Nacional de Medicina Genómica, México, Distrito Federal, Mexico.
3
Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del I.P.N, Distrito Federal, Mexico.
4
Instituto Nacional de Medicina Genómica, México, Distrito Federal, Mexico. Electronic address: jiperez@inmegen.gob.mx.

Abstract

γ-Glutamyl transferase (GGT) is useful as a marker in pathological conditions, including several types of cancer. We optimized the histochemical detection of GGT to assay the gene expression profiles of phenotype-specific cells selected by laser capture microdissection (LCM). For optimization, we used the livers of rats subjected to hepatocarcinogenesis. This model induced nodules of hepatocytes and tumors with GGT activity. To obtain sufficient high-quality RNA after histochemistry and LCM, we included an RNase inhibitor and air-dried the tissue sections. This optimization allowed the visualization of GGT activity in situ and a yield of 1.4 to 2.0 μg of total RNA from 15 to 18 mm² of microdissected tissue (20 µm thickness). The average RNA integrity number in GGT-positive tissue, determined by chip-capillary electrophoresis, was 6.9, and the 28S/18S ribosomal RNA (rRNA) ratio was 1.4. The RNAs were processed for the Rat Gene 1.0 ST Array (Affymetrix). Comparable quality control metrics, such as signal intensity and RNA degradation plots, were found between the LCM samples and non-LCM tissue. The increased expression of Ggt1 expected in GGT-positive tissue was confirmed by microarrays and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). This optimization provided a suitable method for whole-transcript analysis of GGT-positive tissue isolated using LCM.

KEYWORDS:

2-acetylaminofluorene; 2AAF; ANOVA; Atherosclerosis; Carcinoma; DEN; DEPC; DNA microarrays; GGT; GMNA; LCM; Liver neoplasms; RIN; RNA; RNA integrity number; Reverse transcriptase PCR; analysis of variance; cDNA; complementary DNA; diethylnitrosamine; diethylpyrocarbonate; laser capture microdissection; mRNA; messenger RNA; qRT–PCR; quantitative reverse transcriptase polymerase chain reaction; rRNA; ribosomal RNA; γ-glutamyl transferase; γ-glutamyl-4-methoxy-2-naphthylamide

PMID:
24269708
DOI:
10.1016/j.ab.2013.11.015
[Indexed for MEDLINE]

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