Single-channel quantitative multiplex reverse transcriptase-polymerase chain reaction for large numbers of gene products differentiates nondemented from neuropathological Alzheimer's disease

Abstract

Effective approaches using array technologies are critical to understand the molecular bases of human diseases. The results obtained using such procedures require analysis and validation procedures that are still under development. In the context of Alzheimer's disease, in which the identification of molecular mechanisms of underlying pathologies is vital, we describe a robust assay that is the first real-time reverse transcriptase-polymerase chain reaction-based high-throughput approach that can simultaneously quantitate the expression of a large number of genes at the copy number level from a minute amount of starting material. Using this approach within the human brain, we were able to quantitate as many as 19 genes at a time with only one type of fluorescent probe. The number of genes included can be considerably increased. Examples of consistent changes in Alzheimer's disease within these 19 candidate genes included reductions in targets related to the dendritic and synaptic apparatus. These changes were specific to Alzheimer's disease when compared with Parkinson's disease cases. We also present comparison data with microarray analysis from the same brain region and the same patients. The high sensitivity and reproducibility of this technology coupled with appropriate multivariate analysis is proposed here to form a biotechnology platform that can be widely used for diagnostic purposes as well as basic research.

Figure 1

Overview of scqmRT-PCR.

Figure 2

Validation steps of scqmRT-PCR. A: Nineteen targets were processed in parallel from the same amount of starting material and a representative example of threshold cycles obtained during the quantitative round of PCR is shown. B: Standard curves related to the 19 target transcripts were constructed derived from the threshold cycles. C: One percent agarose gel run after the quantitation as a demonstration of amplicon specificity. Such gels are run after each experiment. D: Comparison of regular quantitative RT-PCR and scqmRT-PCR. The upper trend line was obtained following a regular quantitative RT-PCR protocol in which 10⁴ copies of starting material were considered as an unknown copy number on the thermocycler settings. The lower standard curve represents the same experiment performed using scqmRT-PCR protocol. The same starting mRNA copy number was obtained using both techniques.

Figure 3

mRNA copy number per μg total RNA comparisons from control, preclinical, and AD cases. A: Seven transcripts showed consistent change between controls (black, left column) and AD cases (right column). Note that the preclinical cases (gray, left column) matched closer to the AD group. AP180, Dynamin, Syntaxin, ICAM5, and CamK2G are related to the dendritic and the synaptic apparatus. EGR1 showed a greater heterogeneity within the control group. B: Eight transcripts displayed heterogeneity in their mRNA copy numbers in the three groups. C: The three transcripts that showed higher homogeneity within control and preclinical cases compared to AD cases.

Figure 4

PCA performed on scqmRT-PCR results. A: Two-dimensional plot constructed based on the entire set of genes (n = 19). Cases clustered according to their disease status and preclinical cases (C, gray) were positioned closer to the AD cases (AD, black) than to the control cases (C, black). B: Same analysis achieved with AP180, PP2CB, Dynamin, Syntaxin, ICAM5, PARG, and CamK2G. This set of transcripts was sufficient to separate control cases from AD cases and the preclinical cases clustered closer to the AD group. C: Relative importance of principal components for the 19 candidate genes. Note that the first two components accounted for 75.5% of the variance among the cases. D: Relative importance of principal components for seven candidate genes including AP180, PP2CB, Dynamin, Syntaxin, ICAM5, PARG, and CamK2G. Here the first two components accounted for 92.1% of the variance among the cases. E: Two-dimensional plot including four PD cases. Two PD cases (underlined) with concomitant AD clustered close to AD cases. The same set of genes as used for B were used for this analysis. F: Representative gel run after the two rounds of PCR. Targets are in the same order as in Figure 2. The two last lanes, without amplicons, are negative controls (no RT).

Figure 5

Comparison of microarrays and scqmRT-PCR. A: Fold changes between control and AD cases measured with either microarray data or scqmRT-PCR showed inconsistencies for several candidates including FKHR, Integrin 5, Oct 3, and PECAM 1. B: scqmRT-PCR two-dimensional plot of principal components constructed with 18 genes that are also present of microarrays. Note that the preclinical cases (C, gray) clustered with AD cases (AD, black). C: Same analysis as for B but based on microarray’s indirect fluorescence index. Here, the two preclinical cases were not discernible from controls despite their AD histological pathology.