Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus

Abstract

The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by realtime qPCR using primers directed against b-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

Laser capture microdissection and single-cell RT-PCR without RNA purification

“…For example, immunohistochemical staining before LCM identified targeted cell types when morphological criteria failed (Fend et al, 1999;Ball et al, 2002;Vincent et al, 2002). The study of smaller numbers of cells often required preamplification of RNA from LCM-captured cells (Goldsworthy et al, 1999;Bonaventure et al, 2002;Mikulowska-Mennis et al, 2002;Ginsberg and Che, 2004) and rapid staining to preserve nucleic acid integrity in order to generate accurate gene expression profiles (Fend et al, 1999;Mojsilovic-Petrovic et al, 2004).…”

Single cell gene expression profiling in Alzheimer’s disease

“…Moreover, select populations of neurons degenerate, whereas others remain intact during the progression of AD. 118,126,129,193,194 Cholinergic basal forebrain neurons of the NB display phenotypic alterations in cholinergic markers and contain tau pathology in subjects characterized as MCI. 138,195,196 Single cell expression profiling was performed to assess the expression levels of the six tau isoforms (MAPT1-MAPT6; FIG.…”

“…149,150 The orientation of amplified RNAs is "antisense," or a novel "sense" orientation, and one round of TC RNA amplification is sufficient for downstream genetic analyses. 117,126,149,150 Terminal continuation RNA amplification is used for many downstream applications, including gene expression profiling, microarray analysis, and cDNA library/subtraction library construction. Synthesized sense TC-amplified RNA can also be used as a template for in vitro protein translation and proteomic applications.…”

Transcriptional Profiling of Small Samples in the Central Nervous System

“…Rather, this method provides a diagnostic test employed on adjacent tissue sections to ensure the likelihood that an individual case has abundant RNA prior to performing expensive expression profiling studies. A more thorough examination of RNA quality can be obtained via bioanalysis (e.g., 2100 Bioanalyzer, Agilent Technologies), which utilizes capillary gel electrophoresis to detect RNA quality and quantitate relative abundance [18][19][20]. Bioanalysis displays the analytical assessment of RNAs in an electropherogram or digital gel format, with relatively high sensitivity.…”

“…The TC primer contains a span of three cytidine triphosphates (CTPs) or guanosine triphosphates (GTPs) at the 3′ terminus [18]. Adenosine triphosphates (ATPs) or thymidine triphosphates (TTPs) do not perform well as constituents of the TC primer [20]. In this configuration, second-strand cDNA synthesis can be initiated by annealing a second oligonucleotide primer complementary to the attached oligonucleotide [18], and can be performed with robust DNA polymerases, such as Taq polymerase.…”