Presentation on theme: "Mut Wt RNA RNA isolation transfection Cultured cell Splicing reporter PCR Reverse Transcription 5 3 cDNA Agarose gel analysis PCR products Ex vivo analysis."— Presentation transcript:
Mut Wt RNA RNA isolation transfection Cultured cell Splicing reporter PCR Reverse Transcription 5 3 cDNA Agarose gel analysis PCR products Ex vivo analysis of splicing assays Outcome of the experiment : a system to analyze the role of RNA binding proteins 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 ?
A B WT LMNA EXON 11 EXON 12 Lamin A Full length mRNA Normal nuclei Mut LMNA EXON 11 EXON 12 Progerin 1824C>T 150 mRNA Progeria nuclei Figure 1: The use of a cryptic 5 splice site is at the origin of the Hutchison Gilford Progeria Syndrome A) 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.
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 DNA Assay for splicing of your minigene reporter +/- 24hours post transfection Incubate 20 min at room temperature A B C D Figure 2: Quick protocol for splicing reporter transfection A)Prepare two solutions, one containg plasmid DNA and one containing Dreamfect Reagent TM (never vortex dreamfect!) B)Combine the two solutions carefully: drop wise addition, mixing by gently pippetting up and down. C)Add the solution to the cells with freshly replaced medium. Rock the tissue culture plate to homogenize. D)Incubate the cells at 37°C in a CO 2 incubator, evaluate splicing of your reporter.
Lamin 12 Lamin intron 11 globin Intron Lamin exon 11 glo exon 1 12 Lamin 12 Lamin intron 11 globin Intron Lamin exon 11 glo exon 1 12 WT Mut AB Figure 3 : Anticipated results A) When transiently transfected in Hela cells, wild type glo-LMNA splicing reporter produces only the full length splicing isoform. On the other hand 1824C>T glo-LMNA reporter shows a very important use of the cryptic splice site, after agarose gel analysis of RT-PCR products (B). Wt Mut 500 bp 400 bp 300 bp
TROUBLESHOOTING TABLE PROBLEMPOSSIBLE CAUSESOLUTION Low transfection efficiency.Insufficient cell proliferation rate. Amount of DNA used is insufficient. Suboptimal Dreamfect TM /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. Dreamfect TM should be used in excess, for oprtimization, maintain a fixed DNA quantity and then vary the amount of Deamfect TM. 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 TRIREAGENT Be careful, in gel degradation is also possible! PCR product not detected on gel. Low transfection efficiency. cDNA synthesis is not successful. Insufficient RNA in reverse transcription reaction. See top of the table RNA 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.