The overall goal of this experiment is to accurately measure pathogen gene expression in vivo in order to shed light on how a pathogen adapts to the infectious environment and causes damage to its host. This technique paved the way for researchers in the field of infectious disease to explore pathogen gene expression dynamics during a real infection on various tissue types and at multiple time points. The main advantage of this method is that it is highly sensitive and extremely reproducible.
This method makes it feasible to quantify pathogen gene expression from tiny amount of tissues from a real-life infection. To begin preparing reagents, add 2-Mercaptoethanol to buffer RLT and mix well. Prepare a 25:24:1 mixture of phenol-chloroform isoamyl alcohol solution.
Label M-type homogenization tubes and chill on ice. Label two milliliter screw cap tubes and add approximately 300 microliters of zirconia beads to each tube. Have the following instruments ready for use, including a tissue dissociator, a bead beater, a tabletop centrifuge with adapters for 50 milliliter tubes and 96 well plates and a photometer.
Remove previously frozen kidneys isolated from C.albicans-infected mice from a 80 degree celsius freezer and put on ice. Add 1.2 milliliters of buffer RLT to each kidney. Then decant each kidney with buffer into an M-type homogenization tube.
The amount of buffer RLT used is empirically determined. Too much buffer RLT will dilute the sample concentration while too little will make the homogenate very viscous and extremely hard to handle. Next, using a tissue dissociator on the preloaded setting RNA 2.01, homogenize the kidneys.
Then, centrifuge at 1000 x G for one minute. Transfer 600 microliters of each homogenate to a screw cap tube containing zirconia beads and save the remaining homogenate on ice. Add 600 microliters of phenol-chloroform isoamyl alcohol to each tube, tightly close the lids, and vortex on the bead beater for three minutes at four degrees celsius.
Centrifuge at 15, 000 G for five minutes at four degrees celsius. Carefully transfer the aqueous phase to a new 1.5 milliliter microfuge tube, adding equal volume of 70%ethanol. Then load onto the spin column and spin at 8, 000 x G.With 700 microliters of buffer RW, wash each spin column once, followed by two washes with 500 microliters of buffer RPE.
Transfer the columns to dry collection tubes and spin one extra minute to remove any remaining liquid. Finally, use 50 microliters of water to elute the RNA. Then use a spectrophotometer at OD 216 animeters to measure the RNA concentration.
To carry out gene expression profiling using digital bar-coding, begin by thawing the reporter code set and the capture code set on ice. Add 130 microliters of hybridization buffer to the reporter code set. Invert to mix and spin down.
Next add 20 microliters of the mix to each of the 12 reaction tubes. Then add 10 micrograms of total tissue RNA to each tube and mix by pipetting. Depending on the infection model, the inoculum size and the pathogen strain used, the percentage of pathogen RNA within total RNA varies from 0 2%Therefore the total amount of RNA needed to be added for each should be optimized empirically.
Now add five microliters of capture code set to each tube. Mix by flipping the tubes and quickly spin down. Then incubate the reactions in a thermal cycler at 65 degrees celsius with a heated lid for approximately 18 hours.
Remove one sample cartridge from 20 degrees celsius and let it warm to room temperature. Remove two reagent plates from four degrees celsius and spin in a table top centrifuge at 670 x G for two minutes. Next, set up the prep station following step-by-step instructions on the screen, selecting the high-sensitivity option.
Then remove the reactions from the thermal cycler and immediately load on the prep station. After running the three-hour high-sensitivity program, remove the cartridge and use clear tape to seal the lanes. Apply mineral oil to the bottom of the cartridge if required.
Then load the cartridge onto the digital analyzer. Set up the digital analyzer following the step-by-step instructions on the screen, selecting the high-resolution scan option. After running the scanning program, download the results or choose to receive them by email and import the raw data into the manufacturer's software.
Analyze the data according to the text protocol. The protocol demonstrated in this video has the unique advantage in that it uses both a capture probe and a report probe to increase the specificity and reduce the noise from the host RNA. As shown here, background raw counts from an uninfected tissue sample were all below 10, while raw counts from two infected tissue samples were all above 10.
Raw counts from two biological samples had very good correlation with an R squared value of 0.945. The platform also provides sufficient dynamic range to encompass natural biological expression levels. Using a probe set specifying 248 environmental response genes, two phases of pathogen gene expression were determined.
An early gene expression response comprises genes with RNA levels significantly different between the inoculum and 12-hour post-infection samples. A late gene expression response was also discovered that comprises genes with RNA levels that are significantly different between the 12-hour point and 48-hour post-infection samples. These results indicate that C.albicans gene expression is dynamically regulated during invasive infection of a mammalian host.
This method is easy and fast. The whole procedure from tissue collection to expressing data requires less than 48 hours. The hands-on time is around four hours for 12 samples.
While this method is developed to capture pathogen gene expression in vivo, the same can also be used to answer host gene expression. Therefore, researchers can get useful information from both sides of an infection simultaneously.
