HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

RNases are commonly present on skin, hair, dust, laboratory surfaces, solutions, etc. Wear gloves and clean bench surface, pipettes etc. thoroughly before use to avoid RNase contamination. Use of nuclease- free pipette tips, tubes, glassware and reagents is strongly recommended. 1. In vitro Transcription using the T7 High-yield RNA Synthesis Kit The DNA template for the transcription reaction is a linearized plasmid containing the Gaussia luciferase gene and a T7 promoter upstream of the coding sequence (Figure 1B). Thaw the components from the T7 High Yield RNA Synthesis kit and keep on ice. For uncapped RNA, set up the reaction at room temperature in the following order: Component Volume (μl) Final Nuclease free water* X   10X Reaction Buffer 2 1X ATP (100 mM) 2 10mM CTP (100 mM) 2 10mM UTP (100 mM) 2 10mM GTP (100 mM) 2 10mM Template DNA * X 1 μg T7 RNA Polymerase Mix 2   Total 20   *Add 1 μg DNA and make up the total reaction volume to 20 μl with nuclease free water. The amount of water to be added will vary based on the concentration of the template DNA. For capped RNA: Set up the reaction at room temperature in the following order: Component Volume (μl) Final Nuclease free water* X   10X Reaction Buffer 2 1X ATP (100 mM) 2 10mM CTP (100 mM) 2 10mM UTP (100 mM) 2 10mM GTP (20 mM) 2 2mM 3´-0-Me-m7G(5′)ppp(5′)G cap analog (ARCA)(40mM) 4 8mM Template DNA* X 1 μg T7 RNA Polymerase Mix 2   Total 20   *Add 1 μg DNA and make up the total reaction volume to 20 μl with nuclease free water. The amount of water to be added will vary based on the concentration of the template DNA. Mix well by vortexing and incubate the reactions at 37 °C for 2 hours in a dry air incubator. Using steps 1.3-1.4, synthesize capped transcripts of Cypridina luciferase (CLuc) from a linearized plasmid containing the CLuc gene. This mRNA will be co-transfected with the capped and uncapped GLuc mRNA for normalization of GLuc expression. 2. Removal of Template DNA The transcription reactions are treated with DNase I to remove the DNA template before proceeding with purification. Add 70 μl nuclease free water to the transcription reactions followed by 10 μl of DNase I reaction buffer. Add 2 μl DNase I to the reactions. Incubate at 37 °C for 15 minutes in a dry air incubator. 3. Column Purification of Capped and Uncapped RNA Purify the capped and uncapped RNA using the MEGAclearKit as per the manufacturer’s instructions (any spin column based RNA purification kit may be used). Quantify the RNA using a NanoDrop Spectrophotometer. As per the manufacturer’s instructions, assess RNA sample quality using the Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer. 4. RNA Capping using the Vaccinia Capping System Take 10 μg of the purified uncapped GLuc mRNA. A standard capping reaction can cap up to 10 μg (100nt or larger) RNA. If a larger amount of RNA needs to be capped the reaction can be scaled up accordingly. Increase the volume to 15 μl using nuclease free water. Heat the RNA at 65 °C for 10 minutes to remove secondary structures. Place on ice for 5 minutes. To set up the capping reaction, add 2 μl 10X Capping Buffer, 1 μl GTP (10 mM), 1 μl freshly diluted S-adenosyl methionine (2 mM) and 1 μl (10 units) Vaccinia capping enzyme to the 15 μl denatured RNA. Mix well by gently vortexing and incubate the reaction at 37 °C for 30 minutes in a dry air incubator. 5. Column Purification of Capped RNA Purify the capped RNA using the MEGAclear Kit (any spin column based RNA purification kit may be used). As per the manufacturer’s instructions, assess RNA sample quality using the Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer. 6. HeLa Cell Transfection using TransIT mRNA Transfection Kit Plate HeLa cells in a 48-well plate in 150 μl Dulbecco’s Modified Eagle’s Medium (DMEM), High Glucose, supplemented with 10% fetal bovine serum (0.7-2.3 × 105 cells/well). Incubate for 16-24 hours at 37 °C such that the cells are 60-90% confluent. Dilute the GLuc (capped and uncapped) and CLuc RNA to 100 ng/μl concentration. Each transfection is done in replicates of 8. Set up 8 eppendorf tubes. To each tube add 1 μl uncapped GLuc RNA, 1 μl capped CLuc RNA (for normalization) and 25 μl of serum free DMEM. Mix well. Set up 8 eppendorf tubes. To each tube add 1 μl capped GLuc RNA (cap analog), 1 μl capped CLuc RNA (for normalization) and 25 μl of serum free DMEM. Mix well. Set up 8 eppendorf tubes. To each tube add 1 μl capped GLuc RNA (Vaccinia), 1 μl capped CLuc RNA (for normalization) and 25 μl of serum free DMEM. Mix well. Add 0.2 μl mRNA Boost Reagent to the first set of 8 tubes and mix. Add 0.2 μl TransIT mRNA Reagent to the first set of 8 tubes and mix. Incubate at room temperature for 2-5 minutes. (Do not incubate longer than 5 minutes). Add the transfection mixture drop wise to the wells containing HeLa cell cultures (refer Figure 3 for plate set-up). Repeat steps 7 through 10 with the 2nd and 3rd sets of 8 tubes. Incubate overnight (16 hours) at 37 °C in 5% CO2. 7. Gaussia Luciferase (GLuc) Assay Assay the supernatant from each well for GLuc activity using the BioLux Gaussia Luciferase Assay Kit as follows: Prepare fresh assay solution by adding 15 μl of BioLux GLuc Substrate to 1.5 ml of BioLux GLuc Assay Buffer (50 μl assay solution required per sample). Prepare enough for samples and for priming luminometer injector (refer to manufacturer’s instructions). Mix well by inverting the tube several times (do not vortex). Luminescence is measured using the Centro LB 960 microplate luminometer from Berthold Technologies. Set the luminometer to 50 μl injection volume and 2-10 seconds integration. Add 10 μl of the cell culture supernatant from each well into a 96-well black plate. Prime the injector with the assay solution and proceed with measurement of luminescence. Values obtained from untransfected wells will be used as negative controls. 8. Cypridina Luciferase (CLuc) Assay Assay the supernatant from each well for CLuc activity using the BioLux Cypridina Luciferase Assay Kit as follows: Prepare fresh reconstituted substrate (100x) according to instructions given in the kit manual. Thaw the BioLux Cypridina Luciferase Assay Buffer and mix well (protect from light). To prepare the CLuc assay solution, add 15 μl of the reconstituted substrate (100x) to 1.5 ml of BioLux Cypridina Luciferase Assay Buffer (50 μl assay solution required per sample). Prepare enough for samples and for priming luminometer injector (refer to manufacturer’s instructions). Mix well by inverting the tube several times (do not vortex). Keep the solution at room temperature for 30 minutes (protect from light). Luminescence is measured using the Centro LB 960 microplate luminometer from Berthold Technologies. Set the luminometer to 50 μl injection, 1-2 seconds delay and 2-10 seconds integration. Add 10 μl of the cell culture supernatant from each well into a 96-well black plate. Prime the injector with the CLuc assay solution and measure luminescence. Values obtained from untransfected wells will be used as negative controls. 9. Representative Results The T7 High Yield RNA Synthesis Kit can produce up to 180 μg uncapped RNA and 40 to 50 μg of capped RNA per 20 μl reaction. When analyzed on the Agilent 2100 Bioanalyzer, good quality, intact RNA should show a single, sharp peak representing the RNA transcript. A broad peak or multiple peaks indicate RNA degradation. It is also important to verify the size of the RNA. RNA transcripts of a longer length than expected may be due to incomplete digestion of the template plasmid DNA. RNA degradation and lower yield are usually a result of contaminants introduced into the reaction from the template DNA. Figure 2 is an example of the electropherogram and gel image obtained after running high quality, intact RNA on the Bioanalyzer. Transfection of HeLa cells with both capped and uncapped GLuc RNA is followed by incubation and assaying cell culture supernatants for luciferase activity. The 5′ cap structure is important for protecting the RNA against exonuclease degradation8 and for promoting translation initiation9 of the mRNA. Therefore, RNA capping is an essential step for transfection experiments. The co-transcriptional (cap analog) method yields approximately 40 μg RNA that is 80% capped. The protocol utilizing Vaccinia capping enzyme caps up to 10 μg RNA (100 nt or larger) per reaction with nearly 100% efficiency. Figure 4 depicts the difference in luciferase expression between cultures transfected with capped and uncapped RNA samples. It can be clearly seen that cell cultures transfected with capped RNA show much higher expression of luciferase as compared to those transfected with uncapped RNA (which show no luciferase activity). In addition, Figure 4 also validates that both capping techniques, using cap analog and using the Vaccinia capping enzyme, successfully produce functional, capped RNA transcripts that can be translated into protein. The two tailed p-value obtained from the t-test was 0.2583 which indicates that there is no significant statistical difference between the luminescence data from the two capping methods. Figure 1A. RNA Synthesis by T7 RNA Polymerase Figure 1B. pCMV-GLuc vector containing GLuc gene and T7 promoter. Figure 2. Bioanalyzer electropherograms and gel- image of capped and uncapped RNA . The X-axis is the size of the RNA in nucleotides (nt) and the Y-axis represents fluorescence units (FU). A) Electropherogram of GLuc mRNA (uncapped) after purification. B) Electropherogram of GLuc mRNA (capped using Vaccinia capping enzyme). C) Electropherogram of GLuc mRNA (capped co-transcriptionally using cap analog). D) Gel-like image generated by the Bioanalyzer. The ‘L’ lane is the RNA ladder, Lane 1 is uncapped GLuc mRNA, Lane 2 is GLuc mRNA capped using the cap analog and Lane 3 is the GLuc RNA capped using Vaccinia capping enzyme. Figure 3. Example of plate set-up for HeLa cell transfection . Each well consists of plated HeLa cells that are 60-90% confluent. The transfection mix consisting of the appropriate GLuc RNA (uncapped, capped with cap analog or capped with Vaccinia capping enzyme), capped CLuc RNA (for normalization), mRNA Boost Reagent and TransIT mRNA Reagent is added to each well. Each transfection is done in replicates of 8 (same colored wells in the figure). Figure 4.GLuc expression in HeLa cells. Purified capped and uncapped GLuc mRNA was transfected into HeLa cells and incubated overnight (16 hrs) at 37 °C. Cell culture supernatants from each well were assayed for GLuc and CLuc activity and luminescence values were recorded. The GLuc luminescence values were normalized to the luminescence values of capped CLuc RNA. * Luminescence from untransfected cells was subtracted from GLuc and CLuc values before normalization. Trademarks BioLux is a trademark of New England Biolabs, Inc. TransIT is a registered trademark of Mirus Bio LLC. MEGAclear is a trademark of Ambion.