The overall goal of this procedure is to monitor transcript decay rates in proliferating and quiescent fibroblasts. This method can help answer key questions in the molecular biology field such as whether changes in gene expression result from changes in transcript decay rate. The main advantage of this technique is that it provides information on transcript decay rate, not just transcript abundance.
To begin the protocol, establish proliferating and quiescent cultures of fibroblasts. Proliferating fibroblasts should be trypsinized regularly. Quiescent cultures can be established by allowing plates to reach confluence with regular medium changes.
Add ActD 150 microliters to prewarmed medium 10 milliliters for all biological replicate cell culture plates. Add ActD at a concentration of 15 micrograms per milliliter of medium. Mix the solution well.
Aspirate the old medium, and add medium containing ActD to the cells. To collect the zero time point sample, gently aspirate off the ActD medium and pipette between five and 10 milliliters of prewarmed PBS onto a 100-millimeter plate, five milliliters of solution for a 100-millimeter plate, two milliliters for a well of a six-well plate, and one milliliter for a well of a 12-well plate. Gently aspirate the PBS.
Then add phenol-guanidine isothiocyanate solution directly to the tissue culture plate. Pipette the phenol-guanidine isothiocyanate solution cell mixture up and down several times to make a homogenous mixture. Collect each time point after the appropriate amount of time has passed, using the method described earlier.
Incubate the samples at room temperature for five minutes to ensure the complete dissolution of RNA protein complexes. Next introduce spike-in controlled transcripts that can be detected with the methodology used. Introduce these controls into each sample at a known quantity and concentration.
Using a pipette, transfer the phenylguanidine isothiocyanate solution mixture to a 2.0-milliliter microcentrifuge tube. Then add 0.2 milliliters chloroform to the phenylguanidine isothiocyanate solution mixture for every one milliliter of phenylguanidine isothiocyanate solution used. Shake the microcentrifuge tube vigorously for 15 seconds and then incubate the chloroform phenylguanidine isothiocyanate.
After incubation, spin the samples at 12, 000 times g for 20 minutes at four degrees Celsius. The sample should separate into three layers. Transfer the aqueous layer to a new 2.0-milliliter microcentrifuge tube using a micropipette and be careful not to disturb the interphase during this process.
Discard the interphase and organic fractions. Then add an equal volume of 2-Propanol to the aqueous fraction and invert the tube 10 times. Add up to one microliter of 20 milligram per milliliter aqueous glycogen per 20 microliters of sample, especially if the yield is expected to be low.
Incubate the samples at room temperature for 10 minutes. After incubation, spin the samples. Ensure that at the end of the spin, the RNA has precipitated to form a while pellet at the bottom of the tube.
With a pipette, remove and discard the supernatant, taking special care not to disturb the white RNA pellet. Add one milliliter of 75%ethanol per one milliliter of phenylguanidine isothiocyanate solution reagent to wash the pellet. Spin the samples at 12, 000 times g for 10 minutes.
After removing most of the supernatant, use a pipette to remove as much ethanol as possible but do not disturb the pellet. Spins the samples to pellet all excess ethanol. The RNA pellet is less compact at this step and tends to move.
After the spin, remove all excess ethanol from the tube without disturbing the pellet. Allow the RNA pellet to air-dry for five minutes. Add 50 microliters of nuclease-free water to the RNA pellet and resuspend the pellet in nuclease-free water.
Treat the samples of one microliter Deanase to remove DNA and then inactivate the Deanase and cations. Store the RNA samples in 2.0-milliliter microcentrifuge tubes at minus 80 degrees Celsius. Quantify the RNA and check the RNA for purity using a spectrophotometer.
After checking the purity, analyze the RNA with a 1%agarose gel to visualize the extend of degradation. Two clear bands representing 18S and 28S rRNA should be clearly visible. If these bands are not visible, the RNA may be degraded.
Next, monitor the transcript abundance in the collect aliquots of RNA compared with the spiked-in internal standard using Real-Time qPCR. Microarrays, RNA sequencing, or northern blots can also be used. Fluorescence intensities over time correspond to gene expression.
While fibroblasts become contact-inhibited, the transcript-encoding Collagen 3A1, a very important gene for creating the extracellular matrix deposited during wound healing becomes more stable and does not decay as rapidly. Don't forget that working with phenol can be extremely hazardous and it is important not to let it touch your skin. After watching this video, you should have a good understanding of how to monitor transcript decay rates in proliferating and quiescent fibroblasts.
Changes in transcript decay rate can be an important mechanism of gene triangulation.
