This protocol describes a cellular Assay for Monitoring Splicing Efficiency, using a adaptable managing reporter. This reporter Assay can be used to study the effects of disease Associated mutations in the Exxon or splice sites, and to design therapy, particular one particles, to improve the recognition of mutant to five point splice sites. The passage in Hela cells aspirate the spent medium and incubate the cells with three milliliters of 0.2 5%trips in containing one millimolar EDTA, at 37 degrees Celsius for three minutes.
After incubation at seven milliliters of fresh DMEM and transfer the cell suspension to a 10 milliliter tube. Centrifuge the cells. Then resuspend the cell pellet in 10 milliliters of fresh DMEM, and plate the cells on a new tissue culture dish at 20%confluence, for transient transsfection's.
Count the hela cells with a clean hemo cytometer slide and prepare a suspension with a density of 2.5 times 10 to the fifth cells per milliliter, seed one milliliter of the cell suspension into each well of a 12 well plate and incubate overnight at 37 degrees Celsius. The following day aspirate the spent medium and add 800 microliters of fresh DMEM with serum to the plate. Prepare the transfection mixes and Vortex them for 15 seconds.
Then centrifuge the mixes and incubate them at room temperature for five minutes. After the incubation add all 200 microliters of the transfection mix into one well of the 12 well hela cell plate to achieve a final volume of one milliliter per well, and incubate the plate at 37 degrees Celsius for 48 hours. The prepared labeling reactions are incubating.
Resuspend the 25 kilo Dalton molecular weight cutoff size exclusion beads by gentle vortexing for 10 seconds. Next transfer 600 microliters of the resuspended beads to a disposable mini column, placed in a 1.5 milliliter collection tube, and return the beads stock to four degree Celsius. Centrifuge the column and discard the flow through them.
Then wash the beads twice by adding 300 microliters of RNA free water to the column. After the second wash, transfer the mini column to a new 1.5 milliliter centrifuge tube, and add the kinase reaction mix to the column. Centrifuge the column and collect the flow through containing the P32 labeled oligonucleotides.
Add two micrograms of RNA extracted from the heli cells into 200 microliter micro centrifuge tubes and add RNA free water to make up the volume to 6.55 microliters. Next add 0.9 microliters a master mix one to each PCR tube containing the RNA samples, and incubate the tubes first at 65 degrees Celsius for five minutes, and then on ice for one minute. Next at 2.55 microliters, a master mix two, to each tube containing the RNA and master mix one, and incubate the tubes at room temperature for 10 minutes.
After the incubation transfer the tubes to a thermal cycler and incubate first at 50 degrees Celsius for 60 minutes, and then at 70 degrees Celsius, for 15 minutes. After the incubation, add 10 microliters of 2X formamide RNA loading dye to each sample, and proceed to separate the fragments by UIA page. Percy DNA synthesis.
Add two micrograms of RNA extracted from the transected hela cells, into 200 microliter microncentrifuge tubes and add RNAs free water to make up the volume to a low 11 microliters. Next, add two microliters, a master mix one to each sample and incubate first at 65 degree Celsius for five minutes, then on ice for one minute. Next, add seven microliters, a master mix two to each tube and incubate the tubes at room temperature for 10 minutes, followed by incubation at 50 degree Celsius for 60 minutes and 70 degrees Celsius for 15 minutes.
Next, for PCR dilute the C DNA reaction to yield a concentration of 100 nanograms per microliter, and transfer one microliter of each CDNA sample to new PCR tubes. Add 10.5 microliters of the master mix to each tube containing CDNA, and perform the PCR. Using a thermocycler After the PCR is complete.
Add 12.5 microliters of 2X formamide DNA loading dye to each tube. Heat the samples at 95 degree Celsius for five minutes, and proceed to perform a UIA page. Pipette five microliters of diluted CDNA into individual Wells of a 96-well qpcr plate triplicate.
Next add 10 microliters of realtime PCR mix to each well, seal the plates with an optical film, and centrifuge to collect the reactions to the bottom of the Wells, then perform the qpcr while collecting the threshold quantification cycle values of target amplicon. Before loading the markers and samples, flush out the Wells with TBE buffer to remove the settled urea. Then load 10 microliters of each sample per well and run the gel at 300 to 500 volts for two to three hours, or until the xylene reaches the bottom.
After electrophoresis remove the glass plates from the electrophoresis apparatus and carefully separate the two plates so that the gel lays flat on either glass surface, then transfer the gel onto a filter paper and cover it with a plastic wrap, using a gel dryer vacuum dry the gel at 80 degrees Celsius for 30 minutes, then place the dried gel in a phosphor imaging cassette for overnight incubation at room temperature. When expressed in hela cells, the slicing reporter dup 51 minnijean expresses the mature full length transcript in which Exxon two is spliced efficiently. The five prime splice site mutations and dup 51 P reduce Exxon two inclusion from 95 to 30%leading to the formation of a shorter transcript.
Co-expression of dup 51 P with U15A SNRNA rescues Exxon two splicing, and restores the formation of the full length transcript. In contrast, co-expression with wild type U1 or U15G variant does not have the similar effect on dup 51P splicing. Detection of U15A variant transcripts, by primary extension uses the U1 seven to 26 oligo nucleotide, which is 20 nucleotides long and base pairs near the adenine at the fifth position of U1 SNRNA.
In the presence of DATP alone, U1 seven through 26 allows incorporating two to three nucleotides for the endogenous wild type U1 SNRNA yielding a 22 or 23 nucleotide long product. For U15A and U15G variants extension terminates after adding a single adenosine producing a 21 nucleotide product. After normalization to U2 SNRNA expression, transfected U1 wild-type U1 5g and U15A variants were expressed at three to five folds over the endogenous SNRNA.
The quality of the extracted RNA is critical for splicing analysis. Therefore, the use of RNA free water, and decontamination of services with RNAs inactivate agents is highly recommended. The report system described here can be applied to study the role of U1 SN RNAs in splicing regulations for reversing effect of flash price splicing mutations for gene silencing using modify U1SN RNAs.
