This method aims to directly and precisely measure the kinetics of RNA synthesis processing and decay in eukaryotic cells culture. The cells in the presence of four thio uridine to metabolically label the newly transcribed RNA, then lyce the labeled cells and purify the total cellular RNA, perform a thiol specific biotin donation to selectively bio aate, the newly transcribed RNA molecules. Next, use streptavidin coated magnetic beads to capture the four thine labeled newly transcribed transcripts away from preexisting RNA, the RNA fractions containing total, newly transcribed and unlabeled.Preexisting.
RNA can then be subjected to downstream analysis, including Q-R-T-P-C-R, microarrays or next generation sequencing Compared to studies on total cell RNA analysis of newly transcribed, RNA provides approximately a tenfold increase in sensitivity for detecting short-term changes in gene expression. In a tion, it facilitates precise measurements of R half lives, and for the first time actually reveals the kinetics of RND processing. Metabolic labeling of newly synthesized transcripts can be successfully applied to the dissect regulatory molecular mechanisms of gene expression.
It can also be applied to other model organisms like ops, oph, and yeast. Start with a detailed plan of the experiment, allowing at least five minutes in between each condition. Thaw the four thio uridine label just before use and aliquot the amount required for each condition into sterile falcon tubes.
For each treatment condition, transfer five milliliters of the cell culture medium from each 10 centimeter culture dish to the tube containing four thiopurine and mix thoroughly aspirate the remaining medium off the cultures. Then add the label containing medium back to the cells and incubate for the experimentally defined time. After removing the cell culture medium, add five milliliters of triol to each plate.
Incubate for five minutes at room temperature for complete cell lysis. Rinse the plate carefully with the added triol and transfer the samples to polypropylene tubes, which are able to withstand the centrifugation at 13, 000 G.The samples can then be stored at minus 20 degrees Celsius or immediately used to prepare total cellular RNA following the triol protocol. Begin with samples of 60 to 80 micrograms of total cellular RNA For the labeling reaction, add one microliter of 10 x biotin buffer to one microgram RNA diluted in seven microliters nuclease free water.
Then add two microliters biotin HPDP in one milligram per milliliter, DMF per one microgram of RNA and mix immediately by pipetting. If biotin precipitates the DMF content can be increased. The final concentration of 40%Incubate the reaction at room temperature for 1.5 hours under rotation.
Next, add an equal volume of chloroform mix vigorously incubate for two to three minutes until the phases begin to separate and the bubbles start to disappear. Centrifuge the samples, then carefully transfer the upper aqueous phase into a new tube. After a second chloroform extraction, add one 10th of volume of five molar sodium chloride and an equal volume of isopropanol.
To precipitate the RNA from the aqueous phase pellet the RNA by centrifugation. Discard the supernatant and wash the pellet once with an equal volume of 75%ethanol after centrifugation, discard the supernatant. Spin briefly and remove residual ethanol with a 200 microliter pipette.
Spin again and remove residual ethanol with a 20 microliter pipette resuspend, the RNA in 50 to 100. Microliters water mixing well by pipetting. Evaluate the RNA quality by electrophoresis to exclude RNA degradation.
Equilibrate three milliliters of washing buffer per sample to 65 degrees Celsius in a water bath. Also denature the biotinylated RNA samples at 65 degrees Celsius for 10 minutes. Then immediately place on ice.
Position the microm max columns into the magnetic stand. Pre equilibrate the Milton e columns with one milliliter room temperature washing buffer. Meanwhile, add 100 microliters of streptavidin beads to the biotinylated RNA and incubate at room temperature for 15 minutes under rotation.
If any of the columns has not started draining, gently press on the top of the column with a gloved finger. Once the flow starts, the columns drain readily, Apply the RNA bead mixtures to the columns. If the unlabeled RNA is to be analyzed, then collect the flow through of unlabeled RNA fraction, then wash three times with 0.9 milliliters of 65 degrees Celsius washing buffer, followed three times with 0.9 milliliters ambient washing buffer.
Next pipette 700 microliters buffer RLT into new two milliliter tubes and place them underneath the columns. Add 100 microliters of freshly prepared, 100 millimolar DTT to the columns to elute the newly transcribed RNA into the RLT buffer. After three minutes, add another 100 microliters of 100 millimolar DTT to the column and collect the EENT into the same tube.
Continue with the RN easy mini loot cleanup protocol as described in the manufacturers in instructions, add 25 microliters of nuclease free water to the column and put the columns in tubes into a centrifuge. After a one minute spin, remove the columns from the tubes and determine the RNA concentrations using a nano drops spectrophotometer. Be aware that spectra photometric measurements of newly transcribed RNA are not very reliable and tend to overestimate the actual RNA content.
To avoid the need to thaw and refreeze RNA before submitting it to a high throughput assay, we recommend preparing CDNA immediately after the newly transcribed RNA is purified. Use 2.5 microliters of the newly transcribed RNA template in a 20 microliters CDNA synthesis reaction following the manufacturer's instructions. Dilute CD NA samples one to 10 and analyze five microliters of CD NA dilution by Q-R-T-P-C-R.
Store the CD NA samples at minus 80 degrees Celsius to determine the minimal four SU concentration required for efficient recovery of newly transcribed RNA purify the newly transcribed RNA following four SU labeling with increasing concentrations of four SU and analyzed by electrophoresis.Here. The recovery of newly transcribed RNA labeled for one hour in primary human fibroblasts increased substantially from 50 to 200 micromolar four su, but then started to plateau. Alternatively, measure the four SU incorporation by dot blot analysis on the biotinylated RNA using a streptavidin horseradish peroxidase conjugate.
The biotin ation efficiency of biotin. HPDP is approximately threefold less than that of Otto acetyl biotin. Thus one in three four su residues in newly transcribed.
RNA is actually biotinylated by biotin HPDP. The signal intensities can also be quantified by comparison with the biotinylated control. DNA oligos recovery of newly transcribed RNA is highly quantitative.
These data derived from Agilent Bioanalyzer show that the newly transcribed RNA contains significantly greater amounts of large unpled transcripts. Finally, the incorporation rates of four su in newly transcribed RNA can even be directly quantified by spectra photometric analysis. This requires precipitating the newly transcribed RNA with glycogen and isopropanol and ethanol.
The additional peak at 330 nanometers reflects the incorporation rate of four SU into newly transcribed RNA. Don't forget that working with tril can be extremely hazardous. So an antidote for phenol bones consisting of polyethylene glycol 300 mixed with industrial methylated spirits, 70 to 30 should be readily available.
So after watching this video, you should have a good understanding of how to metabolic label and purify newly transcribed RNA, which all in all will take you about eight hours for six to 12 samples.
