1. Analysis der qRT-PCR
The qRT-PCR results for the target gene BACE1 are depicted in Figure 5. The relative fluorescence RFU (Y axis) is plotted against the number of cycles (X axis).
To enable comparison between qRT-PCR runs, a standard threshold was set where a fluorescence signal exceeds background fluorescence. To control for variability in amplification due to differences in starting mRNA concentrations, ACT was used as an internal standard. The relative expression of target mRNA was computed from the target Ct values and the ACT Ct value(∆Ct).
Higher amounts of target mRNA give a good indication of the increased expression of target genes. Relative expression was calculated as per 2-∆Ct.
Figure 5: qRT-PCR results for BACE1
The results obtained for the target genes in control cells were compared to those in transfected cells. The BACE1 level in control cells was set to 100%, the results obtained from the transfected cells are shown in relation to BACE1. The BACE1 results from two groups of students are presented below.
2. Results
Two out of three student groups obtained a complete set of data, including control values. In the first group, 24 hours after transfection with miRNA-107, the BACE1 level decreased to 55.2%; in the second group, it decreased to 36.4%compared to untransfected controls (see Figure 6).
In pilot experiments, RNA was extracted 72 hours after transfection with miRNA-107. In these cells, the BACE1 level went down to 15.7% relative to untransfected controls (see Figure 7).
Figure 6: BACE1 levels in untransfected control cells and transfected cells, 24 hours after transfection with miRNA-107, as obtained by groups 1 and 2
Figure 7: BACE1 levels in untransfected control cells and transfected cells, 72 hours after transfection with miRNA-107, as obtained in pilot experiments
The transfection with miRNA-107 did not considerably influence the APP mRNA level in our SH-SY5Y cells. These results are not shown, nor are the qRT-PCR results from miRNA.
BACE1:
BACE1 is an enzyme which cleaves the precursor protein APP. It thereby gives rise to the neurotoxic β-amyloid peptides which form the senile plaques.
Transfection:
Transfection is the process of introducing foreign DNA or RNA into a eukaryotic cell. We introduced synthetic miRNA-107 mimic into SH-SY5Y cells and screened for changes in the expression patterns of target genes such as BACE1.