Transcriptional Profiling of Small Samples in the Central Nervous System

Abstract

RNA amplification is a series of molecular manipulations designed to amplify genetic signals from small quantities of starting materials (including single cells and homogeneous populations of individual cell types) for microarray analysis and other downstream genetic methodologies. A novel methodology named terminal continuation (TC) RNA amplification has been developed in this laboratory to amplify RNA from minute amounts of starting material. Briefly, an RNA synthesis promoter is attached to the 3′ and/or 5′ region of cDNA utilizing the TC mechanism. The orientation of amplified RNAs is "antisense" or a novel "sense" orientation. TC RNA amplification is utilized for many downstream applications, including gene expression profiling, microarray analysis, and cDNA library/subtraction library construction. Input sources of RNA can originate from a myriad of in vivo and in vitro tissue sources. Moreover, a variety of fixations can be employed, and tissues can be processed for histochemistry or immunocytochemistry prior to microdissection for TC RNA amplification, allowing for tremendous cell type and tissue specificity of downstream genetic applications.

Reduction of β-amyloid and γ-secretase by calorie restriction in female Tg2576 mice

“…Two oligonucleotide primers were used for cDNA synthesis, a poly d(T) primer and a TC primer, which included a sequence that is antisense to the T7 bacteriophage transcriptional promoter. During in vitro transcription, 33 P-UTP was incorporated into newly synthesized RNA species complementary to the cDNA templates (Alldred et al, 2008, 2009; Ginsberg, 2008). …”

“…For qPCR, immunoblot, ELISA, and IHC analysis, we report significant results with p<0.05. Statistical procedures for custom-designed microarray analysis have been described in detail (Alldred et al, 2014; Ginsberg, 2008; Ginsberg et al, 2006). Multiple arrays (n=3–5) were assayed per subject and nested to each subject for increased statistical stringency.…”

“…Signal intensities were compared with negative control arrays, which were hybridized with TC reaction products that contained no initial input RNA. A global normalization procedure effectively minimizes variation due to differences in specific activity and absolute quantity of individual probes (Ginsberg, 2005, 2008; Hemby et al, 2003; Kacharmina et al, 1999). In this analysis scheme, an expression profile of relative changes in mRNA levels was enabled.…”

Expression profile analysis of hippocampal CA1 pyramidal neurons in aged Ts65Dn mice, a model of Down syndrome (DS) and Alzheimer’s disease (AD)

“…qPCR was performed on microdissected CA1 sections from an independent second cohort of animals (Alldred et al 2008; Ginsberg 2008; Alldred et al 2012) containing the hippocampal CA1 region from 10-24 month old Ts65Dn and 2N mice. Taqman qPCR primers (Life Technologies) were utilized for qPCR (Table II).…”

Methods and Compositions for Amplification and Detection of microRNAs (miRNAs) and Noncoding RNAs (ncRNAs) Using the Signature Sequence Amplification Method (SSAM)

“…For the same reason, the present approach eliminated the need to enrich or purify miRNAs prior to miRNA detection. Quantification can be performed through direct assessment of sequence abundance or through quantification of label incorporation (flourescent, radioactive, and biotin-conjugated probes can be employed) similar to our protocols designed for mRNA amplification, notably terminal continuation (TC) RNA amplification [41, 48, 54]. …”