The protocol aims to identify proteins dysfunction leads to changes in the number of mitochondrial DNA molecules. This research may also reveal the factors involving the distribution of mitochondrial DNA within the mitochondria. Mitochondrial replication and maintenance mechanisms are still not fully understood.
Much less is known about regulating the distribution of mitochondrial genomes within organelles and the proteins involved in it. As a result, identifying and characterizing some new players will be of great importance. The main advantage of the technique is its high-throughput and high-content protocol.
We can simultaneously test many experimental conditions and measure various parameters during a single experiment. In genome wide studies, the proposed protocol provides an opportunity to outline a global picture of how nuclear-encoded genetic information regulates its mitochondrial counterpart. Additionally, it has the potential to identify proteins whose dysfunction causes mitochondrial DNA stress and activates the interferon response pathway.
To begin, prepare a 140 nano molar mix of small interfering RNA, or siRNA, by diluting the siRNA in Opti-MEM medium. Add the five microliters of the diluted mix to the wells of a black 384 well cell culture microplate. Prepare the appropriate amount of RNAiMAX transfection reagent solution in Opti-MEM medium and add 10 microliters to each well.
Of the 384 well plate with a reagent dispenser. To begin the cell transfection, add 20 microliters of the HeLa cell suspension to the 384 well plate wells containing diluted siRNA to seed 700 HeLa cells per well. After one hour of incubation at room temperature, place the well plate in an incubator for 72 hours at 37 degrees Celsius and 5%carbon dioxide.
To incorporate BrdU in the transfected cells, add 10 microliters of 90 micromolar BrdU solution to each well after 56 hours of siRNA transfection. Incubate the cells for 16 hours at 37 degrees Celsius and 5%carbon dioxide. The specificity of BrdU incorporation into the mitochondrial DNA of HeLa cells was studied using row zero cells lacking mitochondrial DNA.
To begin the mitochondria labeling, prepare a 20 micromolar solution of BrdU in DMEM with 10%fetal bovine serum, or FBS. Then add the mitochondria tracking dye to adjust the final concentration to 1.1 micromolar. After 15 hours, add 10 microliters of the mitochondria tracking dye solution to the HeLa cell suspension containing wells.
Incubate the cells for one hour. The conditions used to label newly synthesized DNA molecules with BrdU allowed the detection of BrdU labeled DNA in the mitochondria of HeLa cells. The signal obtained with anti BrdU antibodies was specific and only observed in cells treated with BrdU regardless of BrdU treatment, no anti-BrdU or anti-DNA antibody signal was detected in the mitochondria of row zero cells.
Quantifying the fluorescent signal from the anti BrdU antibodies indicated that row zero cells treated with BrdU showed the same low level of fluorescence as the BrdU untreated cells, while the signal for the parental lines A549 and HeLa were 50 fold higher. To begin the HeLa cell fixation, rinse the HeLa cell containing 384 well plate two times with 100 microliters of PBS using the microplate washer. Leave 25 microliters of PBS in the well after the second wash and add 25 microliters of an 8%formaldehyde solution.
Incubate the plate in the dark at room temperature for 30 minutes. Then rinse each well four times with 100 microliters of PBS and leave 25 microliters of PBS in the well after the last wash. Add 25 microliters of 6%bovine serum albumin or BSA in PBS to each well and incubate the plate in the dark for 30 minutes.
To begin the addition of the primary antibody, aspirate the bovine serum albumin, or BSA, from the fixed and blocked transfected HeLa cells, leaving 10 microliters of BSA solution in the well. Then add 10 microliters of the primary antibody solution prepared in 3%BSA in PBS and incubate the plate in the dark at four degrees Celsius overnight. After the incubation, rinse each well four times with 100 microliters of PBS, leaving 10 microliters after the last wash.
Next, add 10 microliters of secondary antibody solution to the 384 well plate. Use isotype-specific antibodies conjugated to fluorochromes, such as Alexa Fluor 488 and Alexa Fluor 555. After incubating the plate in the dark for one hour, rinse each well four times with 100 microliters of PBS and leave 50 microliters of PBS in the well after the last wash.
The use of anti DNA antibodies allowed the monitoring of the mitochondrial DNA distribution in the cell under the given experimental conditions. The downregulation of mitochondrial transcription factor A, TFAM, led to drastic changes in the mitochondrial DNA distribution with fewer mitochondrial DNA spots than in control cells. Quantifying the mean fluorescent signal from the anti-DNA antibody showed a severalfold increase upon TFAM silencing compared to the other conditions tested.
To begin the imaging of transfected HeLa cells, select a sufficiently long exposure time for the individual fluorescence channels in live view mode based on the intensity histograms generated by the imaging software. Set the appropriate number of planes for imaging on the Z axis. Select the appropriate number of fields of view to display per well.
To start the quantitative analysis of acquired images, perform background correction for all the images from all the fluorescence channels. Depending on the size of the analyzed objects, select the appropriate filter size. Next, start the image segmenting by creating a mask of the main object based on the ratio of the intensity of the fluorescence signal to the background for the channel corresponding to the cell nuclei.
Create masks for the subobjects representing BrdU and mitochondrial DNA spots using the fluorescence channels appropriate for the given structures. Set the parameters and measure the intensity of the individual pixels within each mask for all the fluorescence channels. To obtain the total fluorescence intensities for the BrdU or mitochondrial DNA channel, sum up the intensities of all the subobjects assigned to a given cell nucleus.
To obtain the mean fluorescence intensity, divide the total fluorescence intensity by the sum of the area of the subobjects assigned to a given cell nucleus. The imaging results were verified by measuring the mitochondrial DNA and gene expression levels using quantitative real-time PCR. Treatment with dideoxycytidine, or ddC, resulted in a very strong decrease in the mitochondrial DNA levels.
A weaker effect was observed in TFAM and twinkle helicase silencing, while the transfection of cells with mitochondrial DNA polymerase gamma, or POLG siRNA, had a modest effect on the mitochondrial DNA copy number. Quantifying the TFAM and twinkle helicase expression confirmed an efficient reduction in their mRNA expression to approximately 15 to 20%of the control levels. In POLG, the mRNA expression was reduced only to 30%which explains the unexpected modest effect on the mitochondrial DNA copy number.
