Establishment of a protocol for the gene expression analysis of laser microdissected rat kidney samples with affymetrix genechips.
Laser microdissection in conjunction with microarray technology allows selective isolation and analysis of specific cell populations, e.g., preneoplastic renal lesions. To date, only limited information is available on sample preparation and preservation techniques that result in both optimal histomorphological preservation of sections and high-quality RNA for microarray analysis. Furthermore, amplification of minute amounts of RNA from microdissected renal samples allowing analysis with genechips has only scantily been addressed to date. The objective of this study was therefore to establish a reliable and reproducible protocol for laser microdissection in conjunction with microarray technology using kidney tissue from Eker rats p.o. treated for 7 days and 6 months with 10 and 1mg Aristolochic acid/kg bw, respectively. Kidney tissues were preserved in RNAlater or snap frozen. Cryosections were cut and stained with either H&E or cresyl violet for subsequent morphological and RNA quality assessment and laser microdissection. RNA quality was comparable in snap frozen and RNAlater-preserved samples, however, the histomorphological preservation of renal sections was much better following cryopreservation. Moreover, the different staining techniques in combination with sample processing time at room temperature can have an influence on RNA quality. Different RNA amplification protocols were shown to have an impact on gene expression profiles as demonstrated with Affymetrix Rat Genome 230_2.0 arrays. Considering all the parameters analyzed in this study, a protocol for RNA isolation from laser microdissected samples with subsequent Affymetrix chip hybridization was established that was also successfully applied to preneoplastic lesions laser microdissected from Aristolochic acid-treated rats.
Stemmer
Kerstin
K
Environmental Toxicology, Faculty of Biology, University of Konstanz, PO Box-X918, Jacob-Burckhardtstrasse 25, D-78457 Konstanz, Germany. kerstin.stemmer@uni-konstanz.de <kerstin.stemmer@uni-konstanz.de>
Ellinger-Ziegelbauer
Heidrun
H
Lotz
Kerstin
K
Ahr
Hans-J
HJ
Dietrich
Daniel R
DR
D000818:Animals; D034341:Aristolochic Acids; D015925:Cryopreservation / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D007680:Kidney Neoplasms / Q000139:chemically induced / Q000235:genetics* / Q000473:pathology; D007834:Lasers; D008297:Male; D042282:Microdissection; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis; D011230:Precancerous Conditions / Q000139:chemically induced / Q000235:genetics* / Q000473:pathology; D025341:Principal Component Analysis; D012313:RNA / Q000302:isolation & purification; D020871:RNA Stability; D051381:Rats; D011922:Rats, Mutant Strains; D015203:Reproducibility of Results; D013194:Staining and Labeling; D013997:Time Factors; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics
D034341:Aristolochic Acids; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D012313:RNA; C000228:aristolochic acid I