Presentation on theme: "Reverse Transcription Ex vivo analysis of splicing assays"— Presentation transcript:
1Reverse Transcription Ex vivo analysis of splicing assays MutWtRNARNA isolationtransfectionCultured cellSplicing reporterPCRReverse Transcription5’3’cDNAAgarose gel analysisPCR productsEx vivo analysis of splicing assaysOutcome of the experiment : a system to analyze the role of RNA bindingproteins on alternative splicing in cultured cells.Questions that can be answered: what are the factors that regulate a splice site selection ?Where are the regulatory sequences that are responsible for a splicing event ?
2A B WT LMNA Lamin A Full length mRNA Mut LMNA Progerin D150 mRNA EXON 11EXON 12Lamin AFull length mRNANormalnucleiMut LMNAProgerin1824C>TD150 mRNAProgeriaFigure 1: The use of a cryptic 5’ splice site is at the origin of the Hutchison Gilford Progeria SyndromeA) In normal individuals, a distal 5’ splice site is used in exon 11 of LMNA gene. The encoded mRNA will be translated into prelamin A. This protein will undergo 4 steps of postranslational maturation. Mature lamin A is a component of the nuclear envelope .B) In HGPS patients, there is an activation of a cryptic splice site in exon 11 of LMNA gene. The use of this cryptic splice site leads to the deletion of 150 nucleotides, thus 50 aminoacids, from prelamin A. This truncated prelamin A will undergo an incomplete maturation and remain farnesylated, thus aggregating in the nuclear periphery. The nuclear abnormalities that are caused by the truncated protein, called progerin, are responsible for progeria.
3A B C D Assay for splicing of your minigene reporter 0.5 to 2 µg of plasmid DNA per well (in a 6 well plate)2 to 4 µl of dreamfect per µg of plasmid DNAAssay for splicing of your minigene reporter+/- 24hours post transfectionIncubate 20 min at room temperatureABCDFigure 2: Quick protocol for splicing reporter transfectionPrepare two solutions, one containg plasmid DNA and one containing Dreamfect ReagentTM (never vortex dreamfect!)Combine the two solutions carefully: drop wise addition, mixing by gently pippetting up and down.Add the solution to the cells with freshly replaced medium. Rock the tissue culture plate to homogenize.Incubate the cells at 37°C in a CO2 incubator, evaluate splicing of your reporter.
4A B Wt WT Mut Lamin 12 Lamin exon 11 bglo exon 1 intron 11bglobinIntronLamin exon 11bglo exon 1WTMutABFigure 3 : Anticipated resultsA) When transiently transfected in Hela cells, wild type bglo-LMNA splicing reporter produces only the full length splicing isoform. On the other hand 1824C>T bglo-LMNA reporter shows a very important use of the cryptic splice site, after agarose gel analysis of RT-PCR products (B).Wt500 bp400 bp300 bp
5TROUBLESHOOTING TABLE PROBLEMPOSSIBLE CAUSESOLUTIONLow transfection efficiency.Insufficient cell proliferation rate.Amount of DNA used is insufficient.Suboptimal DreamfectTM/DNA ratio.Bad quality of the transfected DNA.Optimize the confluency according to the cells used.It is also possible to transfect freshly seeded cells.High passage number.Mycoplasma contamination.The amount of DNA used can be increased.DreamfectTM should be used in excess, for oprtimization, maintain a fixed DNA quantity and then vary the amount of DeamfectTM.Prepare DNA with cesium chloride gradients or commercial columns.RNA not detected on gel.Degradation of RNA during TRIREAGENT isolation.Verify that the solutions, tubes and tips you use are RNase free.Wear clean gloves at all times.Reduce incubation time with TRIREAGENTBe careful, in gel degradation is also possible!PCR product not detected on gel.cDNA synthesis is not successful.Insufficient RNA in reverse transcription reaction.See top of the tableRNA secondary structure may prevent RT, verify you correctly denature your RNA by incubating at 65°C for 10 min and then cooling on ice for at least 3 minutes.Verify that your sample is not contaminated with DNA or proteins (A260/A280), because this might overestimate the amount of RNA you use for reverse transcription.PCR product generated in the RT minus control.DNA contamination in the RNA sample.Perform a DNase treatment of your RNA (we use RQ1 DNase).Prefer exon junction primers for your PCR reaction.The volume of TRIREAGENT used might have been insufficient for a proper sample homogenization.