Time course infections of hela cells using vaccinia virus begin by setting up the viral infections, infecting the cells, and then harvesting the RNA from the infected cells at several time points post infection. This RNA can later be amplified for hybridization to microarrays for analysis of gene expression. Hi, I am Judy Ann from the laboratory of Kate Irvins at the Whitehead Institute for Biomedical Research.
Today we will show you a procedure for a time course infection with the vaccinia virus in mammalian cells and the subsequent RNA extraction from the infected cells at different time points post-infection. This technique is a beginning step in a larger protocol aimed to extract and amplify RNA in order to assess virus's gene expression during infection, which will complete in two other JoVE videos. So let's get started To set up for the infection, grow hela cells and flasks and wait until the cells are approximately 80%confluent.
Next, be sure to prepare enough viral growth medium for the experiment. Using regular DMEM with 2%FBS and no added antibiotics, the infection can be performed with either recruited stock of vaccinia virus with a known titer or sucrose purified virus with a known titer. If you're concerned about host gene expression, if you are using sucrose purified virus, skip to the next session.
If you are using a crude vaccinia virus, stock sonicate and aliquot of the virus just before infection, and a cup sonicate with a bath of mostly ice and a little water sonicate around 30 W for 20 seconds. Vortex the tube and repeat the sonication three times adding more ice if needed to keep the cup packed with ice and chilled, be sure to vortex in between each sonication step. If you do not possess a cup ator, mix an equal volume of the crude virus stock in point 25 milligrams per milliliter.
Trypsin vortex vigorously incubate the virus stock trips and mix in a 37 degrees Celsius water bath for 30 minutes. Vortex at five to 10 minute intervals to infect cells. First, calculate the amount of viral particles needed to infect the monolayer at the desired multiplicity of infection.
Or MOI. Typically a high MOI between five and 10 is used for infection time courses. Next, add the desired amount of sucrose purified virus or tryp crude virus to 37 degrees Celsius viral growth media and mix well.
You should use enough media to just cover the bottom of the flask. 10 milliliters of media would be enough for a T 1 75 flask. Remove the media from the cells and rinse with room temperature.PBS.
Add the virus or viral growth media to each flask for a zero hour mock time point. Add the viral growth media only with no virus added swirl gently and tilt the plates incubated 37 degrees Celsius in a 5%CO2 incubator for one hour. Be sure to tilt and swirl the plates every 15 minutes to spread the virus uniformly and keep the cells moist.
After incubation. Remove the media containing the virus and rinse three times with room temperature.PBS. Add the maximum amount of viral growth medium to each flask.
For example, 30 milliliters of media for a T 1 75 flask. You can start counting this as your zero hour time point at the desired time point.Post-infection. Check the cells under a microscope and note any cytopathic effects or CPE.
Remove the media and rinse the cells with 30 milliliters of room temperature PBS. Next, add 15 milliliters of trypsin to the cells and incubate for two to five minutes. At 37 degrees Celsius, check under a microscope for cell detachment and tap the flask gently to dislodge cells.
Transfer the cells with a sterile serological pipette into a 50 milliliter conical tube. Wash the flask with an equal volume of cell growth media and add the media to the trypsin cells in the conical tube. Centrifuge the cells at 500 Gs for five minutes at room temperature.
After centrifugation, remove the tryin media from the cell pellet resuspend, the cell pellet, and either triol reagent or the lysis buffer from your desired RNA isolation kit. If you are using triol, divide the sample into one milliliter quats and 1.5 milliliter labeled eend dwarf tubes and freeze negative 80 degrees Celsius. Repeat for all time points.
Harvesting one flask per time point. At this point, your sample should be resuspended and triol reagent and ready to be processed. Add 200 microliters of the BCP phase separation reagent.
For every one milliliter of triol in each tube, BCP or one bromo three chloro propane is used in place of phenol. To reduce genomic DNA contamination you may need to transfer the sample to a larger tube. If you are starting out with a large amount of triol vortex or shake vigorously and incubate room temperature for two to three minutes after incubation, centrifuge the sample at 12, 000 GS for 15 minutes after centrifugation.
An acqui phase should be visible as a clear layer on top. Transfer the acquiesce phase to a fresh tube. You should be recovering approximately 600 microliters for every milliliter of triazole you started out with.
Do a second phenol chloroform extraction. This time using 500 microliters of chloroform per one milliliter triazole instead of BCP. The second chloroform extraction is used to remove any traces of organic solvent from the extraction as even trace amounts can inhibit subsequent amplification steps.
Vortex or shake vigorously, then incubate a room temperature for two to three minutes. Centrifuge the sample at 12, 000 GS for 15 minutes after centrifugation. Transfer the acquiesce phase to a fresh tube.
Remember, the aqueous phase is the clear layer on top. Next, add 20 micrograms of linear acrylamide to each sample. The linear acrylamide acts as a carrier and helps to precipitate the RNA.
Add 500 microliters of isopropanol per one milliliter of triol you started out with to each sample and mix. Well incubate the samples at room temperature for 10 minutes after incubation centrifuge and a micro centrifuge at maximum speed for 10 to 15 minutes. The RNA pellet will be visible at the bottom of the tube after centrifugation.
Very carefully, remove the supernatant from the tube either with a vacuum aspirator or manually by pipetter. Do not disturb the pellet. Wash the pellet with one milliliter of 70%ethanol and centrifuge at top speed for five to seven minutes.
Very carefully. Remove the ethanol from the tube either with a vacuum aspirator or manually by pipetter. Discard the supernatant.
Check that the pellet is visible at the bottom of the tube. Repeat this wash step after the supernatant is discarded. Mark where the pellet is on the tube.
Air dry the pellet for no longer than five minutes. Do not over dry or the RNA will be difficult to reconstitute. Next, if DNA's treatment is planned resus, suspend the pellet in 17 microliters of nuclease free water.
If D'S treatment is not planned, resus suspend in 20 microliters. For D'S treatment, use the KaiGen RNA free DNA set. Add two microliter buffer, RDD and one microliter reconstituted RNAs free.
DNAs one to the resuspended, RNA mixed gently incubated 37 degrees Celsius for 30 minutes. Next, add two microliters of 2.5 millimolar EDTA and incubate at 65 degrees Celsius for five minutes. To inactivate the DNA, do not exceed the inactivation time or temperature as longer times and higher temperatures can cause degradation of the RNA.
Now you have completed the DNA's treatment. Check the RNA concentration by spectrophotometer. Traces of phenol or BCP can be detected as a spike at 270 nanometers beyond the standard peak at 260 nanometers.
When measuring the absorbance of total RNA after extraction, if such contamination appears, retract the RNA using a filter or column-based RNA extraction method before proceeding with amplification. You can store the RNA sample at negative 80 degrees Celsius. You can check the RNA quality if you desire, by using an Agilent bioanalyzer or by running the sample on a denaturing gel and checking the OD readings on the bioanalyzer.
Good RNA quality is indicated by the RNA integrity number or RIN. The RIN is based on a numbering system from one to 10, with one being the most degraded profile and 10 being the most intact. We've just shown you how to perform a time course infection with the vaccinia virus and how to extract the RNA from infected cells at various time points post infection.
When doing this procedure, it's important to remember that there are several critical steps in setting up a synchronous vaccinia infection. First careful sonication or trypsin ization of the virus. It's important to disaggregate virus particles since vaccinia is highly prone to aggregating, which would prevent an even infection of cells.
Second, in order to achieve asynchronous infection, a high MOI of greater than two should be used to ensure each cell is infected. A mixture of infected and uninfected cells will lead to multiple rounds of infection, heterogeneous mixtures of time points, and asynchronous viral, and host transcriptional responses. Third, the infection should be carried out in minimal amounts of media to allow maximum absorption of the virus onto the cells.
In addition, regular shaking of the flask or culture dishes every 10 minutes enables distribution of the virus across the flask and ensures that the cells do not dry out. So that's it. Thanks for watching and good luck with your experiments.
Be sure to watch the other videos in this series where we show the procedure for linear amplification of RNA samples and the preparation and cleanup of fluorescently labeled RNA.
