Laser Capture Microdissection-Generated Target Sample for High-Density Oligonucleotide Array Hybridization

Abstract

Current advances in biomolecular technology allow precise genetic fingerprinting of specific cells responsible for the pathogenesis of human diseases. This study demonstrates the feasibility of generating target samples from laser capture microdissection (LCM) tissues suitable for hybridization of high-density oligonucleotide arrays for gene expression profiling. RNA was successfully isolated by LCM from three paired specimens of oral cancer and linearly amplified using T7 RNA polymerase. Evaluation of the cDNA revealed that five of five cellular maintenance transcripts are detected. Biotinylated cRNA was generated and hybridized to the human Test 1 GeneChip probe arrays, which demonstrated that the RNA is of sufficient quality and integrity to warrant further analysis. Subsequent hybridization of the samples to the HuGenFL GeneChip probe arrays revealed that 26.5%-33.0% of the approximately 7000 represented genes are expressed in each of the six samples. These results demonstrate that LCM-generated tissues can generate sufficient quality cRNA for high-density oligonucleotide microarray analysis, an important step in determining comprehensive gene expression profiling using this high-throughput technology.

Expression Profiling of Ductal Carcinoma in Situ by Laser Capture Microdissection and High-Density Oligonucleotide Arrays

“…To determine whether LCM-derived material could be used in oligonucleotide array expression studies, we first investigated the reliability of an approach using transcription-based linear amplification of RNA, 7,8,10,14 starting with 10 ng of total RNA. This quantity was chosen because we have previously shown that 10 ng of highquality RNA can be obtained with minimal LCM dissection (MAW, unpublished data).…”

Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis

“…The biotinylated cRNA from the 10 samples (normal and cancer) were further used to hybridize the A ymetrix Test-1 probe arrays to determine cRNA quality and integrity. The arrays contain probes representing a handful of maintenance genes and a number of controls (Ohyama et al, 2000). Analysis of the arrays con®rmed the RT ± PCR ®ndings.…”

“…Most of these patients have a history of smoking and alcohol consumption, which are the major etiological causes of oral cancer (see Materials and methods). The quality and quantity of isolated RNA was examined by reverse transcription polymerase chain reaction (RT ± PCR) of ®ve cellular maintenance gene transcripts of high to low abundance (glyceraldehyde-3-phosphate dehydrogenase; tubulin-a; b-actin; ribosomal protein S9; uniquitin C) (Ohyama et al, 2000). The quantity of isolated RNA was also assessed with RiboGreen RNA Quantitation Reagent and kit (Molecular Probes, Eugene, OR, USA) using spectro¯uorometry (Bio-Rad, Hercules, CA, USA).…”

“…These procedures were carried out as previously described (Ohyama et al, 2000). Normal mucosa was obtained from the contralateral side of the patient's oral cavity.…”

cDNA Array Hybridization after Laser-Assisted Microdissection from Nonneoplastic Tissue

“…The T7-based linear amplification 8,9 was the first approach to be shown to generate a representative mRNA profile that allows to combine microdissection with array technology. 10,11 As the linear amplifi-cation is very time consuming and susceptible to various disturbances and even failures leading to RNA loss and degradation, a PCR-based technique was suggested.…”