The overall goal of this procedure is to isolate the total RNA of Pseudomonas aerogen recovered from murine ECMs. This is accomplished by first harvesting the sequel luminal contents. Next, the bacterial RNA is isolated.
Then D'S treatment and RNA cleanup are performed. Finally, the RNA is quantified and the purity of the isolate is checked by bioanalyzer and spectro photometric analysis. Ultimately, bacterial RNA of sufficient quality and quantity can be obtained for subsequent experimental evaluation such as by QPCR, analysis, transcription profiling, or RNA sequence experiments.
The main advantage of this technique over existing methods like triol extraction, is that this method incorporates multiple lysis steps in extremely rigorous acid, female chloroform extraction steps to ensure high quality RN.A visual demonstration of this method is critical as the lysis and extraction steps are difficult to learn because written protocols emit small details that are critical for success. Demonstrating the procedure will be Eduardo Lopez Medina, a postdoctoral fellow, and Megan Neubauer the lab manager, both from my laboratory. Before beginning the harvest, IMSA cleans stainless steel mortar in a liquid nitrogen bath.
Then after euthanizing a pseudomonas aerogen colonized six to eight week old C3 HHEN mouse place the animal in a supine position on a styrofoam board and shower the abdomen with 95%ethanol. Next, make a midline longitudinal incision through the skin from the sternum to the perineum. Then draw back the skin and peritoneum and expose the abdominal cavity.
Now resect the entire cecum using forceps. Hold the secum over the stainless steel mortar and then snip both ends of the secum with dissection scissors. Fill a P 1000 pipette tip with one milliliter of sequel flush eight buffer, and then insert the tip into the proximal end of the cecum.
Flush the sequel flush eight buffer and the sequel luminal contents into the stainless steel mortar. Now grind the sequel. Flush eight contents with a sterile pestle.
Then pour ethanol into a container of dry ice and place a 50 milliliter polypropylene conical tube into the dry ice ethanol bath. Transfer the ground frozen sequel luminal contents into the conical tube and store the tube at minus 80 degrees Celsius. This next series of steps are the trickiest of the procedure.
Given the complex nature of materials recorded from the MI cecum, the repeated cold filoform extractions are absolutely required in order to achieve acceptable or inequality and integrity between three to five extractions may be required before the wide interface between the aquas and organic phases is eliminated. Transfer one volume of acid phenol chloroform into a 50 milliliter Oak Ridge centrifuge tube. Secure the cap with parfum and then warm the tube in a 65 degree Celsius water bath.
Place half a volume of lysis buffer into a 50 milliliter polypropylene conical tube, and then boil the buffer for five minutes. After removing the sequel luminal contents from minus 80 degrees Celsius storage, briefly warm the tube with a brisk hand rub and then add the boiling lysis buffer to the tube. Shake the mixture to homogenize the lysis buffer's SQL luminal content solution.
Boil the lysis buffer and SQL luminal contents for five minutes. With periodic vortexing unseal the Oak Ridge centrifuge tube containing the phenol chloroform solution and transfer the SQL contents lysis sample to the tube. Then reseal the cap with perfil and place the tube back in the 65 degrees Celsius water bath for 10 minutes for texting the tube every two minutes.
Wash the sample at 2, 500 times G for 15 minutes at four degrees Celsius. Now carefully transfer the aqueous phase to a fresh 50 milliliter Oak Ridge centrifuge tube, avoiding any of the white interface. Then add an equal volume of acid phenol chloroform to the new tube.
Seal the cap with perfil and mix the contents well by vortexing the tube at high speed. Continue centrifuging the sample and transferring the aqueous phase to the centrifuge tube. If the volume of the upper aqueous phase becomes too small, it can be transferred to a smaller diameter tube.
Continue this process until there is no visible white interface between the aqueous and organic phases. When the white interface is gone, carefully transfer the aqueous phase to a fresh centrifuge tube. Then add an equal volume of chloroform, isoamyl alcohol to the tube, seal the cap and mix the contents well by vortexing.
Next, after centrifuging the sample again, transfer the aqueous phase to a 15 milliliter polypropylene conical tube and add an equal volume of isopropanol. Then incubate the solution at minus 20 degrees Celsius for at least two hours or overnight. After incubating the sample, centrifuge the tube for 45 minutes.
After identifying the gel-like pellet at the bottom of the tube, remove the supernatant. Now wash the pellet with one milliliter of ice cold, 70%ethanol by vortexing the tube, and then centrifuge the sample at 10, 000 times G for five minutes at four degrees Celsius. Finally, remove the supernatant and invert the tube to let the pellet air dry at room temperature for 10 minutes.
Then resuspend the RNA pellet in 200 microliters of RNAs free water. First, add 20 microliters of 10 x turbo, DNA buffer and two microliters turbo DNAs to the micro fuge tube and mix the solution gently. Then incubate the tube at 37 degrees Celsius for 20 minutes, and then add 20 microliters of resuspended, DNA inactivation reagent to the tube and mix.Well.
Now incubate the tube for five more minutes at room temperature with occasional mixing. Next, spin down the pellet in a micro centrifuge at 10, 000 times G for 1.5 minutes, and then transfer the supernatant to a new micro fuge tube. Add one volume of cold acid phenol chloroform to the pellet, and then mix the solution thoroughly by vortexing for one minute.
After centrifuging the solution for two minutes at 12, 500 times G, transfer the aqueous phase to a new tube and add one volume of chloroform isoamyl alcohol. Then mix the solution by vortexing after centrifuging the solution again, under the same conditions. Transfer the aqueous phase to a new micro fuge tube.
Then add 0.1 volume of three molar sodium acetate and 2.5 volumes of ice cold, 100%ethanol. Vortex the tube to mix. Incubate the sample at minus 80 degrees Celsius for 30 minutes.
Then after centrifuging at 12, 000 times G for 30 minutes at four degrees Celsius, remove the supernatant. Finally wash the pellet with one milliliter of ice cold, 70%ethanol. Remove the supernatant and air dry the pellet for 10 minutes.
Then resuspend the pellet in 100 microliters of RNAs free water. The Agilent Bioanalyzer is a microfluidics based platform for sizing, quantification, and quality control of D-N-A-R-N-A proteins and cells and utilizes an RNA integrity metric known as RNA integrity number or R-I-N-R-N-A. Extracted with this protocol produces two 60 to two 80 ratios ranging from 1.7 to 2.0 and RIN values greater than or equal to 7.0.
An example of an Agilent bioanalyzer electropherogram of the bacterial RNA is shown here on the left, and a gel-like image of pseudomonas arosa recovered from murine SQL contents is shown here on the right. Following this procedure, other methods like quantitative PCR or RNA sequencing can be performed in order to answer additional questions like refined gene expression profiling.
