Shuttling of cDNA constructs into appropriate expression vectors
cDNA construct from the RZPD clone library were integrated into expression vectors us-ing commercially available Gateway® technology (Invitrogen). The entry clones of FADD (RZPDo839C12160) and DDX24 (RZPDo839H0780) were shuttled into the destination vec-tors pTL-HA1-D48, pc-myc-CMV-D12, pPAReni-DM and pFireV5-DM as well as in pdEYFP-Amp and pdECFP-pdEYFP-Amp according to the shuttling protocol of the Gateway® system (Invitrogen;
www.invitrogen.com).
Chemical transformation of E. coli
Plasmid DNA was transformed into chemically competent E. coli Mach1-T1R. 5µl of plasmid DNA was added to 50 µl of competent Mach1-T1R cells and incubated on ice fro 30 min followed by heat-shock for 30 sec at 42°C. Subsequently, the cells were put back on ice for 1 min to cool down, mixed with 250µl S.O.C. medium without antibiotics and then incubated at 37°C under shaking for 1 hour. Aliquots were then plated onto antibiotics treated LB-agar plates and grown at 37°C over night.
Plasmid preparation from E. coli
For amplification of plasmid DNA, LB medium supplemented with the appropriate antibiotics was inoculated with E. coli colonies carrying the desired plasmid and grown over night at 37°C in an incubator-shaker, and subsequently harvested by centrifugation. Depending on the DNA quantities plasmid DNA was purified using the Qiagen Plasmid Mini or Midi Kit according to manufacturer’s instructions.
Determination of DNA and RNA concentration
To determine the concentration of nucleic acids, the absorbance of the purified DNA or RNA in aqueous solution was measured at 260 and 280 nm in a NanoDrop spectrophotometer (Peqlab).
An A260 reading of 1.0 is equivalent to ~50 µg/ml double-stranded DNA or ~40 µg/ml single-stranded RNA. The A260/A280 ratio is used to assess DNA/RNA purity. An A260/A280 ratio of 1.8 - 2.1 is indicative of highly purified DNA or RNA.
Restriction digest of DNA
Restriction digests were performed in the restriction buffers and according to the protocols sup-plied by the manufacturer. For restriction digestion ca. 1 µg of the plasmid preparation were in-cubated at 37°C for 1-2 h. The reactions were stopped by adding 10x DNA sample buffer and the products were separated by electrophoresis on an agarose gel.
DNA electrophoresis
To separate DNA fragments the samples mixed with loading buffer were loaded to a 1 % (w/v) aga-rose gel, which contained 0.5 g/ml ethidium bromide to visualize the DNA after separation under UV-light, as a running buffer 1x TBE was used.
RNA isolation from mammalian cell culture and mouse brain tissue
Total RNA from mammalian cells was extracted and purified using RNeasy Mini Kit (Qiagen) fol-lowing the manufacturer’s instructions. For disruption of the cells the cell pellets were resuspended in lysis buffer provided in the kit and spin through QIAShredder homogenizer columns (Qiagen).
To avoid contamination of the RNA preparation with chromosomal DNA samples were incubated with DNaseI after binding to the RNeasy spin column using the RNase free DNase Kit (Qiagen).
The purified total RNA was eluted from the columns using 30 µl RNase-free water.
To isolate total RNA from mouse brain I used the RNeasy Lipid Tissue Mini Kit (Qiagen) accord-ing to the manufacturer’s protocol. Samples derived from mouse striatal tissue were mechanically homogenized in QIAzol Lysis Reagent (Qiagen) and mixed with chloroform. After addition of chloroform, the homogenate was separated into an aqueous and an organic phase by centrifuga-tion. RNA partitions to the upper, aqueous phase, while DNA partitions to the interphase and proteins to the lower, organic phase or the interphase.
The upper, aqueous phase was extracted and ethanol was added to provide appropriate binding conditions. The samples were then applied to the RNeasy spin column, where the total RNA binds to the membrane and phenol and other contaminants are efficiently washed away. Total RNA was finally eluted in 60 µl RNase-free water.
The concentration of the total RNA preparations was determined using a spectrophotometer (NanoDrop8000; Peqlab) as described above.
cDNA synthesis via reverse transcription
Using the retroviral enzyme reverse transcriptase one can transcribe RNA into complementary cDNA. Such as other polymerases this specific RNA dependent DNA polymerase needs an oli-gonucleotide bound to the RNA and serving as the starting point of the strand
complementa-the cDNA syncomplementa-thesis from cellular total RNA complementa-the RevertAid™ H minus first strand cDNA syncomplementa-the- synthe-sis Kit (Fermentas) was used according to the manufacturer’s protocol. For reverse transcription and cDNA production 1 – 2 µg of total RNA isolated from mammalian cells or mouse brain was used.
Quantitative real-time PCR
The quantitative real-time PCR (qRT-PCR) is a highly sensitive technique to simultaneously amplify and quantify a DNA target sequence, based on the principle of a normal PCR (Higuchi et al., 1992).
The DNA quantification occurs by fluorescence measurement during the PCR-cycles. For fluores-cence detection either the PCR products can be labelled with fluorescent dyes which unspecifically intercalate into DNA double-strands or a fluorescence probe is bound to the DNA template. The latter one, known as “TaqMan probe” is a sequence specific oligonucleotide, carrying a fluorophore (“reporter”) at their 5‘-end and a quencher of fluorescence at the 3‘-end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5‘ to 3‘ exonuclease activity of the Taq polymerase breaks the reporter-quench-er proximity and thus allows unquenched emission of fluorescence, which can be detected aftreporter-quench-er excitation with a laser (Lee et al., 1993). The intensity of the fluorescence signal is direct propor-tional to the amount of amplified double stranded PCR product and therefore increases over time of the PCR reaction.
To perform a qRT-PCR, cDNA obtained from the reverse transcription of total RNA (see previ-ous paragraph) was diluted 1:1000 in RNAse-free H2O and added to the PCR reaction as template.
For detection of endogenous DDX24 mRNA purchased primer/probe mixes specific for either rat or mouse DDX24 were used (TaqMan Gene Expression Assay DDX24; FAM-labelled; Applied-Biosystems). As endogenous controls for normalization of the relative DDX24 mRNA levels to the amount of total RNA I used the TaqMan GAPD (GAPDH) Endogenous Control Assay (rat or mouse specific) and the TaqMan ACTB Endogenous Control Assay (rat specific), respectively.
All used probes of the endogenous controls are VIC-labelled (AppliedBiosystems). For detection of Htt103Q-EGFP or Htt25Q-EGFP mRNA a self designed primer/probe set was used (FAM-labelled; Chapter 4.1.6, Table 4.1). All real-time PCR reactions were performed in triplicates as multiplexed samples detecting cDNA of both, target and endogenous control within the same well (possible because of their different fluorescence labelling). The composition of the real-time PCR reactions is shown below in Table 4.3a/b.
Table 4.3a: Components of a multiplexed real-time PCR reaction using purchased TaqMan Gene Expression Assays
Component Volume (µl)
cDNA template 5
TaqMan Gene Expression Assay DDX24 (20X) 1
TaqMan Endogenous Control Assay (20X) 1
TaqMan Universal PCR Master Mix (2X) 10
Nuclease-free H2O 3
Final volume 20
Table 4.3b: Components of a multiplexed real-time PCR reaction using self designed primer/probe set
Component Volume (µl)
cDNA template 5
forward primer (10 pmol/µl) 0.5
Reverse primer (10 pmol/µl) 0.5
Fluorescent probe (10 pmol/µl) 0.5
TaqMan Endogenous Control Assay (20X) 1
TaqMan Universal PCR Master Mix (2X) 10
Nuclease-free H2O 2.5
Final volume 20
All real-time PCR reactions were performed in the Applied Biosystems 7500 Real-Time PCR System using the standard amplification program:
50 cycles 50°C 15min
95°C 2min 95°C 15sec 60°C 45sec
}
The quantification was done by analysis of the data collected during the run. The data was anayzed by the so called comparative threshold cycle method (CT). The CT-value corresponds with the PCR cycle in which the fluorescence intensity detected in the sample overcomes the background signal (threshold) for the first time. The more target cDNA is present in the sample the earlier the threshold will be exceeded, resulting in a small cycle number and a low CT-value. The CT-value of the sample is then normalized to the CT-value of its endogenous control (calibrator) giving its ΔCT-value, calculated as:
ΔCT = CT target - CT calibrator
To further compare the samples of interest with the corresponding negative controls a second comparison/calculation step is performed in which the ΔCT-value of the sample is normalized to the ΔCT-value of the corresponding control (for example induced PC12 cells vs. non induced PC12 cells) giving its ΔΔCT-value, calculated as:
ΔΔCT = ΔCT sample - ΔCT control
On basis of the ΔΔCT-value the x-fold change of target gene expression can be calculated using the formula:
-ΔΔCT
x-fold change = 2