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6b. Materials and Methods
1. Cell culture
The human brain cells (SH-SY5Y),which served as an in vitro model to study AD, were purchased from Sigma-Aldrich. Cell culture work was carried out using the tissue culture hood in the school lab. Laminar air flows protect cell cultures from contamination.
Cells were cultivated in cell culture flasks (75 cm2).
For passaging, cells were washed in PBS buffer and then incubated with 1x trypsin/EDTA in PBS for 90 seconds at 37°C.
After trypsinization, the required amount of cell suspension was transferred into a new cell culture flask containing
12 mL of media (10% DMEM und Ham’s F-12, 1:1).
For transfection, cells were trypsinized and transferred into a reaction tube (15 mL) containing 2 mL of media. The cell suspension was centrifuged at 400 g for 5 minutes. Media was removed and the pellet was resuspended in fresh media. The number of cells per mL was determined.
2. Transfection

For each transfection run, cells were plated at a density of 100.000 cells / 2 mL in one well of a 12-well plastic tissue culture plate. An amount of 37.5 ng of miRNA-107 mimic was suspended in media (0% FBS) and 3 μl HiPerFect Transfection Reagent (Qiagen) were added. After 10 minutes of incubation at room temperature, 100μL of this solution were added to each well. Transfection was allowed to occur for 24 hoursand 72 hours, respectively.

Subsequently, RNA was isolated according to the protocol outlined below.
3. RNA isolation
RNA was isolated from transfected and untransfected control cells using the iScript RT-qPCR Sample Preparation Reagent Kit (Bio-Rad). Media was removed from the wells and the cells were washed using PBS buffer. Subsequently, 100 of µL iScript RT-qPCR Sample Preparation Reagent were added to each well. Tissue plates were moved gently and then incubated for 5 minutes at room temperature. The suspension was then transferred into reaction tubes which were kept at –20°C before reverse transcription was carried oute.
4. Reverse Transcription
4.1. Introduction
In reverse transcription, DNA is generated from an RNA template. This process is catalysed by the enzyme “reverse transcriptase”. Reverse transcription contradicts the central dogma of molecular biology. According to this dogma, biological information flows from DNA to proteins: DNA is copied into mRNA in a process called “transcription” and proteins are synthesized using the mRNA as a template in a process referred to as “translation”. However, reverse transcription occurs in viruses, such as HIV, and also eukaryotes.
In the present study, we isolated mRNA of genes of interest and copied the mRNA molecules into cDNA using reverse transcriptase. After the synthesis of complementary single-stranded cDNA molecules, the RNA molecules are degraded by RNase H. Once this process is completed, second strand synthesis starts which gives rise to double-stranded cDNA molecules.
4.2. Reverse Transcription from mRNA
The students worked in three groups. Each group isolated RNA from transfected cells and untransfected control cells.

For the production of cDNA with mRNA as a template, we used the iScript Reverse Transcription Supermix for RT-qPCR (Qiagen). First, we thawed the iScript Reverse Transcription Mix and the RNA templates on ice. Then, we mixed 12 µL nuclease-free water and 6 µL 5x iScript Reverse Transcription Supermix with 2 µL RNA template in a reaction tube (0.5 mL).The solution was incubated at different temperatures in the Thermocycler (Bio-Rad). After 5 minutes of priming at 25°C, the solution was incubated at 42°C for 30 minutes. Subsequently, the solution was incubated at 85°C for 5 minutesto inactivate the reverse transcriptase. The samples were then kept –20°C until further use.

The aim of the subsequent steps was to assess the relative expression of our target genes, notably BACE1 and APP, using qRT-PCR. To control for variability inamplification due to differences in starting mRNA concentrations, β-ACT, a housekeeping gene, was used as an internal reference

4.3. Reverse Transcription from miRNA
To produce cDNA with miRNA as a template, we used the miScript II RT Kit (Qiagen) according to the manufacturer’s instructions. Briefly, we mixed 8 µL 5x miScript HiSpec Buffer, 4µL RNase-freewater and 4 µL 10x Nucleics Mix with 4 µL miScript Reverse Transcriptase Mix in a reaction tube. Subsequently, 10 µL of this solution were transferred in a reaction tube and 10 µL of the RNA template from transfected cells and untransfected control cells were added. The solution was incubated for 60 minutes at 37°C, followed by 5 minutesat 95°C in a Thermocycler (Bio-Rad). Upon completion of the programme, the solution was kept at –20°C.
5. qRT-PCR for target genes
5.1. Principles of PCR
The polymerase chain reaction (PCR) is one of the most important techniques in biomedical research. It allows the amplification of a DNA fragment across several orders of magnitude within a short time frame. A PCR reaction encompasses three steps. First, the DNA is denatured at a high temperature (about 95°C), yielding single-stranded DNA molecules. Second, the reaction temperature is lowered to allow the binding of the DNA primers to the single-stranded DNA template. PCR primers are short oligonucleotides, which serve as a starting point for DNA synthesis.

A pair of primers, a “forward” and a “reverse” primer, is hybridized to the target DNA to determine the region to be amplified in the PCR process (see Figure 1). Finally, the DNA polymerase synthesises a DNA strand complementary to the template at a temperature assuring maximal enzyme activity. The three steps are depicted in Figure 2. On average, these steps are repeated about 40 times to amplify the target DNA 1012-fold. Successful PCR requires the following reagents and components: DNA template, dNTPs (deoxynucleoside triphosphates), two primers, DNA polymerase with a temperature optimum at around 70°C and the cofactor magnesium.

PCR is carried out to assess the activity of target genes. Specific primers determine the target genes and exclusively allow them to be amplified. The more target DNA is available, the fewer PCR cycles are required.
Figure 1: PCR primers are oligonucleotides thatserve as starting points for DNA synthesis. PCR primers allow the target DNA to be determined. For successful PCR, a “forward primer” and a “reverse primer” are required, binding each to one strand of the originally double-stranded DNA.
Figure provided by Michael Gadermaier, 2011
Figure 2: The PCR cycle. First, the two DNA strands are separated (“denaturation”). Second, the primers bind to the single-stranded DNA templates (“annealing”). Third, DNA polymerase synthesizes DNA using the primers as starting points (“elongation”). Subsequently, the cycle is repeated.
RealTime PCR Applications Guide, Bio-Rad (2006)
5.2. qRT-PCR
The quantitative Real-Time PCR (qRT-PCR) is a technique that allows the amplification and simultaneous detection of DNA. In contrast to PCR, the detection of amplified DNA material does not occur at the end of the process but real time, as the process progresses. Fluorescent dyes such as SYBR Green intercalate with the double-stranded PCR products and allow quantification (see Figure 3). The green light emitted by the DNA-dye-complex is recorded and enables the determination of the relative amount of target genes: The fewer PCR cycles required to obtain a certain fluorescence signal, the higher the amount of target genes and the higher the activity of these genes.
Figure 3: SYBR Green binds to double-stranded DNA and emits green light as blue light is shined on it. The fluorescence signal peaks at the end of the elongation step.
RealTime PCR Applications Guide, Bio-Rad (2006)
5.3. Protocol
For qRT-PCR, we used the qRT-PCR Sso SYBR Green Supermix (Bio-Rad). Primers for screening BACE1, APP and ACT were provided by Eurofins. We mixed 5 µL Supermix with 2.5 µL nuclease-free water and 2 µL cDNA. qRT-PCR was carried out using the CFX96 Real-Time PCR Detection System(Bio-Rad).
Prior to the actual PCR cycle, the solution was heated to 95°C. The temperature was held for 30 seconds before denaturation commenced. The steps of the PCR cycle, depicted in Table 1, were repeated 40 times. At the end of the programme, a melting curve was set up as a functioning test (see Table 1).
Step Temperature Time Cycles
Activation 95°C 30 sec 1
Denaturation 95°C 5 sec 35 bis 40
Elongation 60°C 40 sec 35 bis 40
Melting cuve 65°C bis 95°C (in Steps of 0,5°C) 2 bis 5 sec per Step 1
Table 1: Steps of qRT-PCR for target genes
5.4. Samples
Our cell culture was divided into two parts. One part was not transfected and served as a control whereas the other part was transfected with miRNA-107. We extracted mRNA from both and transcribed the mRNA molecules into cDNA using reverse transcriptase. We then carried out qRT-PCR using the cDNA as a template. We screened for BACE1 and APP; ACT, a housekeeping gene, served as an internal control. Eurofinsprovided the primer pairs for the genes that we tested. The comparison of the cDNA and therefore mRNA content between the transfected cells and the untransfected controls would allow a conclusion on the effect of miRNA-107 on the expression of the genes of interest.
6. qRT-PCR for miRNAs
6.1. Principles of qRT-PCR for miRNAs

The quantitative Real-Time PCR for miRNAs follows the same principles as the qRT-PCR does for target genes (see 6.2.5.).
A poly(A) tail is added to the 3’-end of the cDNA molecules in the reverse transcription reaction, which serves as a docking point for the oligo(dT) primer.

Figure 4: -PCR using miScript SYBR® Green PCR Kit and miScript Primer Assay (Qiagen) (3)
In the first PCR cycle, a specific miRNA primer binds to miRNA-107 and thereby excludes the amplification of other miRNAs (1). To synthesize the complementary strand, a miRNA universal primer [2] was used (see Figure 4).
In the present study we compared the miRNA-107 level in transfected cells to untransfected controls. According to Wang et al. (4), the miRNA-107 level is decreased in early AD. We hypothesized that untransfected SH-SY5Y cells would exhibit low miRNA-107 levels and that these levels would be increased after transfection. The transfection should therefore also impact the expression pattern of genes regulated by miRNA-107 (see 6.2.5.).
6.2. qRT-PCR protocol for miRNAs
The qRT-PCR was carried out using the miScript SYBR® Green PCR Kit and the miScript Primer Assay for hsa-miRNA-107 (Qiagen) according to the manufacturer’s instructions. The miScript SYBR® Green PCR Kit kit contains QuantiTect SYBR® Green PCR Master Mix and miScript universal primer (1). The specific primer for miRNA-107 was provided by Qiagen (5). Reagents and cDNA samples were thawed on ice. The reagents were mixed in PCR reaction tubes (Bio-Rad (6)) as outlined in Table 2. The samples were analysed in triplets.
1 reaction µl 3 reaction µl
SYBR Green PCR Mastermix 5.00 15.0
cDNA template (cDNA: 100ng-100fg) 1.00 3.0
Total 6.00 18.0
Nuclease-free water 2.00 6.0
10x miScript Universal Primer 1.00 3.0
10x miScript Primer 1.00 3.0
Total Primer 4.00 12.0
Σ= 10 30
Table 2: Reagents for qRT-PCR for miRNA-107, optimized after (7)
The qRT-PCR was carried out using the CFX96 Real-Time PCR Detection System (Bio-Rad [8]). The PCR steps are outlined in Table 3 (9).
Step Temperature Time Cycles
Activation Step 95°C 15 minutes 1
Denaturation 94°C 15 seconds 35 to 40
Annealing 55°C 30 seconds 35 to 40
Elongation 70°C 30 seconds 35 to 40
Melting curve 65°C to 95°C (in steps of 0,5°C) 2 to 5 seconds per step 1
Table 3:Steps of qRT-PCR for miRNA-107 (9)
6.3. Literature
(1) Qiagen miScript SYBR® Green PCR Kit Product Details, http://www.qiagen.com/products/miscriptsybrgreenpcrkit.aspx (2.2.12)
(2) Qiagen miScript II RT Kit Product Details, http://www.qiagen.com/products/miscriptiirtkit.aspx#Tabs=t1
(3) Qiagen miScript II RT Kit Product Details, http://www.qiagen.com/products/miscriptiirtkit.aspx#Tabs=t1(external link) Figure: Selective conversion of mature miRNAs into cDNA in miScript HiSpec Buffer
(4) Wang, W. X., B. W. Rajeev, et al. (2008). "The expression of microRNA miR-107 decreases early in Alzheimer's disease and may accelerate disease progression through regulation of beta-site amyloid precursor protein-cleaving enzyme 1." J Neurosci 28(5): 1213-1223.
(5) Qiagen miScript Primer Assays (Hs_miR-107_2 miScript Primer Assay (100), MS00031255), http://www.qiagen.com/products/miscriptprimerassays.aspx#Tabs=t1
(6) PCR Strips Bio-Rad Low-Profile 0.2 ml 8-Tube Strips without Caps #TLS-0801+ Optical Flat 8-Cap Strips #TCS-0803
(7) Qiagen miScript SYBR® Green PCR Kit Resources, Qiagen miScript SYBR® Green PCR Kit used with miScript Primer Assays or miScript Precursor Assays Quick-StartProtocol, Seite 3, http://www.qiagen.com/products/miscriptsybrgreenpcrkit.aspx#Tabs=t2
(8) CFX96 Real-Time PCR Detection System, Bio-Rad
(9) Qiagen miScript SYBR® Green PCR Kit, Resources, Qiagen miScript SYBR® Green PCR Kit used with miScript Primer Assays or miScript Precursor Assays Quick-StartProtocol, Seite 4, http://www.qiagen.com/products/miscriptsybrgreenpcrkit.aspx#Tabs=t2