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Table 2 Validation of microarray analysis by RT-PCR (three independent repeats) of 17 selected genes

From: Gene expression and functional annotation of human choroid plexus epithelium failure in Alzheimer’s disease

  RT-PCR#1 RT-PCR#2 RT-PCR#3 RT-PCR Microarray
Gene F.C. P value F.C. P value F.C. P value Mean F.C. Mean F.C.a P value*
AGXT2L1 21.19 0.003 17.36 0.010 15.57 0.005 18.04 9.45 0.001
SPP1 5.92 0.004 6.59 0.002 8.09 0.001 6.87 4.16 0.040
ITGAV 3.21 0.004 3.20 0.026 3.33 0.011 3.25 3.43 0.002
ZBTB1 2.17 0.022 7.22 0.001 3.51 0.001 4.30 3.39 0.008
IFRD1 b 1.16 0.731      1.16 3.39 0.001
BAMBI 2.26 0.002 1.81 0.007 2.16 0.026 2.08 3.1 0.003
IER5 4.43 0.051 4.58 0.002 4.41 0.011 4.47 2.98 0.003
ARMET 2.64 0.002 2.94 0.001 3.04 0.001 2.87 2.87 0.004
LPIN1 2.41 0.038 2.25 0.011 2.58 0.007 2.41 2.45 0.002
DNAJA4 5.42 0.038 4.63 0.011 4.65 0.011 4.90 2.15 0.021
ATF4 2.31 0.004 2.56 0.002 2.07 0.011 2.32 2.04 0.003
DLC1 3.66 0.001 3.47 0.002 3.47 0.001 3.53 1.93 0.008
ANXA5 −2.39 0.002 −3.15 0.001 −2.99 0.001 −2.84 −2.52 0.033
EPHX2 −3.81 0.001 −2.28 0.001 −2.45 0.001 −2.85 −2.53 0.007
KCNJ13 −2.82 0.004 −2.56 0.007 −2.86 0.002 −2.75 −2.54 0.018
CIRBP −4.72 0.002 −7.12 0.001 −6.84 0.001 −6.23 −2.72 0.003
CLDN5 −8.80 0.001 −5.49 0.001 −4.99 0.001 −6.43 −3.1 0.004
LYPLAL1 −3.82 0.002 −3.81 0.001 −4.22 0.001 −3.95 −3.3 0.002
PRDM16 −6.50 0.002 −4.17 0.001 −3.90 0.001 −4.86 −3.62 0.004
  1. Individual gene expression (and validation). In order to assess the validity of the RNA expression data obtained by microarray, we performed RT-PCR analysis on cDNA prepared from laser-dissected CPE of the same healthy control (Br0–1) and late stage AD samples (Br5–6) as used for the microarray. We essentially followed the procedure of van Soest SS et al. (2007) [33]: In short, RT-PCR validation was done, in triplicate, on a selection of 17 genes. These genes were chosen on the basis of high expression level (>90%), significantly different expression between late stage AD and healthy control (p < 0.05) and high fold change (FC). Next, we searched for unique and efficient primers in the last 1 kb of the 3′ region the mRNA, as this area was used for the design of the oligo’s on the microarray and the microarray amplification procedure employed the poly a tail. We measured three times the expression levels of the resulting set of 17 genes by RT-PCR and calculated three times the FC and the relevant p values. Table 2 summarizes the findings and compares RT-PCRs with the outcome of the microarray. The significant difference as indicated by the microarray was confirmed for 16 of these genes, whereas the 17th gene did not amplify well in our hands in the RT-PCR, and showed unexpectedly a very low expression, perhaps due to limitations in primer design. Pearson’s correlation coefficient of the FC’s was remarkably high (r = 0.95) and significant (p < 0.01), confirming the gene expression results of the microarray. F C: Fold change in mRNA expression of CPE of AD (Braak 5,6) vs. healthy (Braak 0,1) donors. Pearson’s correlation coefficient between F.C. of micoarray and RT-PCR was significant (r = 0.95, p < 0.01). RT-PCR#1, RT-PCR#2, RT-PCR#3 denote three independent RT-PCRs on 7 human control samples and 7 and AD affected samples. *Some genes are represented with multiple probes on the array. If so, the value shown is the lowest P value. aSome genes are represented with multiple probes on the array. If so, the value shown is the mean F.C. bOur RT-PCR showed for this gene unexpectedly a very low expression, perhaps due to primer design