Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

All experiments described were approved by Institutional Review Boards at Princeton University and the University of California, Los Angeles.

1. Prepare Proliferating and Contact-inhibited Fibroblasts for ActD Time Course

NOTE: This protocol uses a timecourse with four timepoints. Three biologically independent samples can be collected per timepoint by collecting different tissue culture plates in one experiment, or the experiment can be repeated multiple times with different cultures of cells. In our experience, one 10 cm (diameter) tissue culture dish (one plate) provides sufficient RNA for analysis. If needed, multiple tissue culture dishes can be pooled for each sample to increase the number of cells at each timepoint. In addition, the same experiment can be performed with fibroblasts isolated from different individuals as truly independent biological replicates.

1. Establish proliferating and quiescent cultures of cells for the transcript decay analysis. For proliferating cells, split the cells every three days while the contact-inhibited cells are becoming contact-inhibited.
   1. For the contact-inhibited cells, change the medium every 2 days for 7 days. Split the proliferating cells 2 days before the contact-inhibited fibroblasts are ready for collection. An example of a cell culture scheme is shown in Figure 1. The remaining steps in this section provide a detailed protocol for establishing and splitting the cells.
2. Isolate primary dermal fibroblasts from neonatal skin according to established protocols¹³. Passage fibroblasts to ensure a homogeneous population.
   NOTE: Fibroblasts can be frozen and thawed, if needed.
3. Thaw or replate fibroblasts, if needed. Grow fibroblasts in DMEM with 10% serum under sterile conditions in a tissue culture incubator at 37 °C and 5% CO₂. Use fibroblasts that have been grown for fewer than 10 passages. Fibroblasts should be <80% confluent on the day of splitting cells to make proliferating and quiescent populations.
4. To split a tissue culture plate into multiple plates, prewarm PBS, trypsin, and medium with serum in a 37 °C water bath for 20 min.
5. Aspirate or use a pipet to remove the medium from each tissue culture plate with cells to be replated.
6. Pipet warm PBS onto each plate (10 mL of solution for a 150 mm plate, 5 mL of solution for a 100 mm plate, 2 mL for a well of a 6 well plate, and 1 mL for a well of a 12 well plate).
7. Aspirate or pipet to remove PBS from each tissue culture plate.
8. Gently pipet 1x Trypsin-EDTA onto each plate (8 mL of solution for a 150 mm plate, 3 mL of solution for a 100 mm plate, 2 mL for a well of a 6 well plate, and 1 mL for a well of a 12 well plate).
   NOTE: Make 1x Trypsin-EDTA by diluting sterile 10x Trypsin-EDTA in sterile PBS. The final 1x solution should be 0.05% trypsin and 0.02% EDTA.
9. Incubate trypsin with cells for 5 min.
10. Gently tap the plate to dislodge cells from the plate.
11. Pipet to resuspend cells into a single cell suspension.
12. Transfer cells in trypsin solution to a conical tube containing the same volume of DMEM with 10% serum (8 mL of solution for a 150 mm plate, 3 mL of solution for a 100 mm plate, 2 mL for a well of a 6 well plate, and 1 mL for a well of a 12 well plate).
13. Spin down cells at 4 °C for 5 minutes at 1,000 x g to remove trypsin.
14. Resuspend cells in an appropriate volume of medium and count with a hemacytometer to determine cell concentration.
15. Seed 5 x 10⁵ cells per 100 mm tissue culture plate for proliferating and contact-inhibited cells.
16. Using this method, establish the proliferating and contact-inhibited samples required for analysis (Figure 1).

2. ActD Time Course

1. Resuspend ActD at a stock concentration of 1 mg/mL (For 1 mg of ActD, use 950 µL of sterile PBS and 50 µL of sterile DMSO).
   NOTE: Frozen stock solutions of ActD should be stable for a month at -20 °C. Dilute solutions should be discarded and not stored for further use.
2. Add ActD to the appropriate volume of prewarmed medium for all biological replicate cell culture plates that were set up for the 0, 120, 240, and 480 min timepoints (Figure 2). Add ActD at a concentration of 15 µg per mL of medium. Mix well, aspirate off the old medium, and add ActD-containing medium to the cells.
   1. For a 1 mg/mL stock, add 15 µL ActD for each mL of medium, e.g., for 10 mL of medium for a 100 mm plate, add 150 µL ActD.
3. To collect the zero timepoint sample, gently aspirate off the ActD medium, and pipet prewarmed PBS onto the cells. Pipet 10 mL of solution for a 150 mm plate, 5 mL of solution for a 100 mm plate, 2 mL for a well of a 6 well plate, and 1 mL for a well of a 12 well plate.
4. Gently aspirate or pipet off the PBS.
   NOTE: All steps involving RNA isolation should be performed with RNase-free water, RNase-free microcentrifuge tubes, and RNase-free pipettes. Gloves should be worn and changed frequently when working with RNA.
5. Add phenol-guanidine isothiocyanate solution (1 mL/10-cm plate with 4 x 10⁵ to 4 x 10⁷ cells) directly to the tissue culture plate.
   NOTE: Phenol-guanidine isothiocyanate solution is a monophasic solution that maintains RNA quality while lysing cells and separating RNA, DNA, and protein from each other.
   CAUTION: Phenol and guanidine isothiocyanate are corrosive. Use appropriate protection.
6. Pipet the phenol-guanidine isothiocyanate solution-cell mixture up and down several times to make a homogeneous mixture.
   NOTE: Phenol-guanidine isothiocyanate solution lysate can be stored at 4 °C overnight or at -20 °C for up to a year.
7. Collect each time point after the appropriate amount of time has passed using the method described in steps 2.3-2.6.

3. Isolation of Total RNA from Phenol-guanidine Isothiocyanate Solution Lysate

1. Incubate samples at room temperature for 5 min to ensure the complete dissolution of RNA-protein complexes.
   NOTE: The steps of the phenol-guanidine isothiocyanate solution protocol can be performed at room temperature unless otherwise noted.
2. Introduce spike-in control transcripts that can be detected with the methodology used. Introduce these controls into each sample at a known quantity and concentration.
   NOTE: The external RNA control consortium (ERCC) spike-in control mix¹⁴ has been specifically designed as a spike-in control for RNA-Seq. It can also be used for Northern blots or real-time qPCR. Spike-in controls specifically designed for microarray technologies are also available (see Table of Materials).
3. Transfer the phenol-guanidine isothiocyanate solution mixture to a 2.0 mL microcentrifuge tube with a pipette. Add 0.2 mL chloroform to the Phenol-guanidine isothiocyanate solution mixture for every 1 mL of phenol-guanidine isothiocyanate solution used (e.g., add 0.3 mL chloroform to 1.5 mL phenol-guanidine isothiocyanate solution mixture).
4. Shake the microcentrifuge tube vigorously for 15 s and then incubate chloroform-phenol-guanidine isothiocyanate mixture at room temperature for 3 min.
5. Spin samples at 12,000 x g for 20 minutes at 4 °C.
   NOTE: After the initial spin, the sample should separate into 3 layers – a red organic layer at the bottom, a white/pink interphase in the middle, and a clear aqueous layer on the top (Figure 3).
6. Transfer the aqueous layer to a new 2.0 mL microcentrifuge tube using a micropipette.
   NOTE: Be careful not to disturb the interphase during this process. It is okay to leave some of the aqueous fraction. It is better to leave some of the aqueous fraction than to also include some of the interphase layers, as including the interphase layer would contaminate the RNA with DNA. The aqueous layer will be approximately half of the initial volume, so about 0.9 mL if the phenol-guanidine isothiocyanate solution/chloroform mixture was 1.8 mL.
7. Discard the interphase and organic fractions.
8. Add an equal volume of 2-propanol to the aqueous fraction and invert the tube 10 times. Add up to 1 µL of 20 mg/mL aqueous solution of glycogen per 20 µL of sample, especially if the yield is expected to be low because fewer than 10⁶ cells were collected.
   NOTE: This will result in a dense, solid pellet that is easy to monitor after the spin steps that follow.
9. Incubate samples at room temperature for 10 min.
10. Spin samples at 12,000 x g for 20 minutes at 4 °C. Ensure that, at the end of this spin, the RNA has precipitated to form a white pellet at the bottom of the tube.
11. With a pipette, remove and discard the supernatant taking special care not to disturb the white RNA pellet.
    NOTE: A loose pipet tip held in hand can be used to remove excess ethanol. If the RNA pellet is disturbed, but not discarded, the investigator can repeat the spin and remove the supernatant again. Avoid discarding the pellet as this will require the investigator to repeat the entire procedure.
12. Prepare a solution of 75% ethanol and 25% nuclease-free water. Add 1 mL of 75% ethanol per 1 mL of phenol-guanidine isothiocyanate solution reagent to wash the pellet.
13. Spin samples at 12,000 x g for 10 min at 4 °C.
14. After removing most of the supernatant, use a pipet to remove as much ethanol as possible, while still taking care not to disturb the pellet.
    NOTE: The RNA pellet is less compact at this step and tends to move. Be extra careful when handling the pellet at this point to avoid losing the RNA.
15. Spin samples 12,000 x g for 2 min at room temperature to pellet all excess ethanol.
16. Remove all excess ethanol from the tube using a micropipette taking care to not disturb the pellet.
17. Let the RNA pellet air dry for 5 min.
18. Add 50 µL of nuclease-free water to the RNA pellet and resuspend the pellet in the nuclease-free water.
19. Treat samples with 1 µL DNase (2 units/µL) to remove DNA and then inactivate DNase and cations (see Table of Materials).
20. Store RNA samples in 2.0 mL microcentrifuge tubes at -80 °C.

4. Analysis of Transcript Abundance

1. Quantify RNA and check RNA for purity using a spectrophotometer.
   NOTE: The ratio of absorbance at 260 nm to 280 nm should be approximately 2.0 for RNA.
2. Analyze RNA with a 1% agarose gel or an equivalent technology to visualize the extent of degradation.
   NOTE: Two clear bands representing 18S and 28S rRNA should be clearly visible (Figure 4). If these bands are not visible, the RNA may be degraded. Trouble shooting tips for RNA degradation are provided in the Discussion.
3. Monitor transcript abundance in the collected aliquots of RNA compared with the spiked-in internal standard using microarrays¹⁵, RNA-Seq¹⁶, real-time qPCR¹⁷, or Northern blots¹⁸.
   1. Determine the abundance for each transcript at each time point compared with a spiked-in control of known abundance.
      NOTE: For microarrays, the values will be fluorescence intensities. For Northern blots, bands on the X-ray film can be quantified. For real-time qPCR, transcript abundance can be determined by comparison with known standards with the 2^-σσC_T method¹⁹. For RNA-Seq, normalized values can be determined as FPKM or fragments per kilobase of exon per million reads mapped. For isoform-specific decay rates, RNA-Seq data can be analyzed using isoform-aware methodologies such as DEXSeq²⁰. Isoform-specific decay rates are best determined with RNA-Seq. If they are to be determined with microarray data, then the data must be analyzed on a probe-by-probe basis rather than a gene-by-gene basis. The analyst must be careful when interpreting information from a limited number of probes. For instance, there may be 5 or fewer probes that are informative about a particular isoform. In this case, the analyst will need to determine whether the behavior of these probes is sufficiently consistent so that the results are reliable.
4. Perform real-time qPCR on the isolated RNA¹⁷ in the collected samples to analyze rapidly decaying genes, like c-Myc and a slowly decaying gene like GAPDH, to gain confidence that transcript decay rates were accurately detected.
   NOTE: For further confirmation, isoform-specific real-time qPCR probes can be developed and used to monitor the abundance of specific isoforms over time²¹.

5. Decay Rate Constant Determination

1. For each timepoint, log-transform the abundance values for each transcript (gene-specific or isoform-specific).
   NOTE: Log-transforming the values will convert exponential decay curves to lines that can be fit with a linear decay model²².
2. Fit the expression profile for each transcript (gene-specific or isoform-specific) to a linear decay model. The formula for such a model is f(t) = C – r*t. In this equation, C is a constant that represents the starting amount of a transcript, r is the rate of decline, and t is the time since the beginning of the experiment. From this equation, determine the decay rate as r*ln(10) (see reference¹¹).
   NOTE: The maSigPro software package is a regression-based approach designed to detect significant gene expression profile differences in time course data²³. Sample code and results for maSigPro are available at: https://www.bioconductor.org/packages/release/bioc/vignettes/maSigPro/inst/doc/maSigProUsersGuide.pdf.
3. Filter out genes that fail to fit a log-linear decay model. This can be accomplished with an ANOVA F-test.
   NOTE:The F test is based on the F-statistic, which is defined as the variation between sample means divided by the variation within samples. Correct the p-values for multiple comparisons by applying the linear step-up Benjamini and Hochberg false discovery procedure²⁴. A q-value greater than 0.05 with the F-test can be used as a cutoff for genes that fail to fit a log-linear decay model.
4. Perform an ANOVA F-test to determine transcripts with a significant interaction term between a categorical cell cycle condition and time (false discovery rate, FDR <0.05).