cDNA Array Hybridization after Laser-Assisted Microdissection from Nonneoplastic Tissue

Abstract

Differential gene expression can be investigated effectively by cDNA arrays. Because tissue homogenates result inevitably in an average expression of a bulk of different cells, we aimed to combine mRNA profiling with cell-type-specific microdissection. Using a polymerase chain reaction (PCR)-based preamplification technique, the expression profile was shown to be preserved. We modified the existing protocol enabling to apply the total amount of extracted RNA from microdissected cells. A mean amplification factor of nearly 1000 allowed to reduce the demand of initial RNA to ϳ10 ng. This technique was used to investigate intrapulmonary arteries from mouse lungs (ϳ500 cell equivalents). Using filters with 1176 spots, three independent experiments showed a high consistency of expression for the preamplified cDNAs. These profiles differed primarily from those of total lung homogenates. Additionally, in experimental hypoxia-induced pulmonary hypertension, amplified cDNA from intrapulmonary vessels of these lungs was compared to cDNA from vessels dissected from

Combination of Erythropoietin and Sildenafil Can Effectively Attenuate Hypoxia‐Induced Pulmonary Hypertension in Mice

“…The animals were exposed to 3 weeks of either hypoxia (10% oxygen) or normoxia (room air), after which the animals received the different drugs for additional 2 weeks under the same environmental conditions. This hypoxic mouse model for PH is well established, has been described by several groups, 20,[24][25][26] and is known to result in PH after 3 weeks of hypoxic exposure. The normobaric hypoxic chamber was opened for 1 hour once per week to replace bedding, water bottles, and chow.…”

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

“…This inevitably results in the averaging of the expression of different cell types and the expression profile of a specific cell type may be masked, lost or ascribed to and dismissed as illegitimate transcription (Chelly et al 1989) because of the bulk of the surrounding cells. Indeed, it is not surprising that significant differences have been detected in the gene expression profiles of microdissected and bulk tissue samples (Fink et al 2002, Sugiyama et al 2002. This is particularly relevant when comparing gene expression profiles between normal and cancer tissue since normal cells adjacent to a tumour may be phenotypically normal, but genotypically abnormal or exhibit altered gene expression profiles due to their proximity to the tumour (Deng et al 1996), and some tumours have significantly larger immune cell infiltrates than others (Bustin et al 2001).…”

Endotoxin-induced gene expression differences in the brain and effects of iNOS inhibition and norepinephrine

“…Porphobilinogen deaminase (PBGD) was used as reference gene. For cDNA synthesis, reagents and incubation steps were applied as described previously [21]. PCR reactions were set up using the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, Karlsruhe, Germany).…”