The overall goal of this procedure is to isolate mitochondria associated with translating mRNAs. This is accomplished by first harvesting yeast cells grown under mitochondria enriching conditions. The second step is to gently lice the cells in the presence of cyclo heide.
Next mitochondria are separated from soluble components by differential centrifugation. The final step is extraction of RNA from mitochondria and control samples. Ultimately, northern analysis is used to show the purity and quality of the mitochondria associated RNA.
The main advantage of this technique is that many types of RNA can be isolated in a single preparation. Therefore, a comparative analysis can be made. This method can help answer key question In cell biology such as intracellular targeting and organal function.
In this procedure, mitochondria will be purified from 100 to 150. Milliliters of yeast cells grown to an OD 600 of one to 1.5 at 30 degrees celsius on a non fermentable growth.Medium. Centrifuge cells at 3000 times gravity for five minutes at room temperature, discard the supernatant and wash the pellet with double distilled water centrifuge again.
After discarding the supernatant weigh the cell pellet. Typically about 0.6 grams is obtained from 100 milliliters of cells. Resus suspend the pellet in one milliliter of DTT buffer per 0.5 grams of cells.
It is important to treat the cells with a reducing agent in order to break the dis sulfide bonds within the cell wall, thereby improving hydrolysis incubate the cells for 10 minutes at 30 degrees Celsius with gentle shaking. Next centrifuge cells at 3000 times gravity for five minutes at room temperature. Discard the supernatant and resuspend the pellet.
In one milliliter of xmal liase buffer per 0.15 grams of cells do not vortex. Measure the OD 600 of a 10 microliter aliquot of the cells in 990 microliters of water and record this value. Add freshly prepared xmal liase to the cells to hydrolyze glucose polymers at the beta one three glucan linkages and generate Sphero plast.
From here onward Sphero plast should be kept in an isotonic solution in order to avoid lysis, incubate cells for 15 minutes at 30 degrees Celsius with gentle shaking. To verify hydrolysis of the cell wall and sphere of plast generation, mix 10 microliters of the cells with 990 microliters of water and measure the OD 600 Sphero plats are expected to lice due to osmotic difference and the OD 600 should be at least tenfold less than the value determined prior to adding xmal liase. If not, continue incubation with xmal liase for another 15 minutes.
Re suspend the PHE of Plast with 100 milliliters of recovery medium and transfer the sphere of plast to an Helen Meyer Flask. Incubate for one hour at 30 degrees Celsius with shaking. This recovery step is necessary since translation is arrested and mRNA localization is disrupted during the XYs treatment.
After one hour add 0.1 milligrams per milliliter, cyclo heide, and transfer the Sphero plast to pre chilled 50 milliliter conical tubes. Cyclo Heide edition is important to freeze the association of ribosomes with mRNAs. Thus, ribosome dependent mRNA Association with mitochondria is maintained.
Centrifuge, the sphere of plass at 3000 times gravity for five minutes at four degrees Celsius. Discard the supernatant and wash twice with cold mannitol. Buffer reus.
Suspend the sphere of plass with four milliliters of cold manitol buffer and transfer the suspension to a down homogenizer of 15 milliliter capacity equipped with a tight fitting pestle. Gently break the sphere of plass with 15 strokes and transfer the lysate to a 13 milliliter tube. Centrifuge the lysate at 1, 500 times gravity for six minutes at four degrees Celsius.
To pellet the nuclei and unbroken cells. Carefully transfer the super natin to a new tube. Set aside one milliliter or 25%of the sample as an unfractionated or total sample.
Transfer 50 microliters of this sample to a new tube and add 15 microliters of four XLSB. For western blood analysis, RNA will be precipitated from the rest of the total sample for northern and microarray analyses. Centrifuge the super natin at 10, 000 times.
Gravity for 10 minutes at four degrees Celsius. To pellet the mitochondria, transfer the super natin to a new tube and keep it on ice. This is the cytosolic fraction.
Transfer 50 microliters of this sample to a new tube and add 15 microliters of four XLSB. For western blot analysis, RNA will be precipitated from the rest of the cytosolic sample for northern and microarray analyses. Wash the pellet with three milliliters of mannitol, buffer and centrifuge again at 10, 000 times.
Gravity for 10 minutes at four degrees Celsius. Reus suspend the pellet with three milliliters of mannitol buffer. This sample contains the mitochondria and is referred to as the mitochondria fraction.
Transfer 50 microliters of this sample to a new tube and add 15 microliters of four XLSB for Western blot analysis. To begin this procedure, add to each sample one volume of eight molar guad dium, HCEL and two volumes of 100%ethanol vortex and incubate for at least two hours at minus 20 degrees Celsius. Centrifuge the samples at 10, 000 times gravity for 20 minutes at four degrees Celsius.
Discard the supernatant. Be careful as the pellet might be unstable. After washing the pellet with 70%ethanol reus, suspend the pellet with 400 microliters of RNAs free water and transfer the sample to a new einor tube.
Precipitate the RNA again by adding 0.1 volume of three molar sodium acetate pH 5.2 and two volumes of 100%ethanol vortex and incubate for at least two hours at minus 20 degrees Celsius. Centrifuge samples at 20, 000 times. Gravity for 20 minutes at four degrees Celsius.
Discard the supernatant and wash with 70%ethanol. Air dry the pellet and resus suspend with RNAs free water store RNA. Samples at minus 80 degrees Celsius.
Samples can subsequently be used for northern or microarray analyses. To prepare the RNA for microarray analysis, first resus suspend each MRNA pellet obtained from guin HCL and ethanol precipitation with 650 microliters of RNAs free water and transferred to a screw cap eend orph tube. Then add 650 microliters of acidic phenol chloroform and vortex vigorously centrifuge at maximum speed for two minutes.
Transfer 500 microliters of the upper aqueous phase to a new tube. Avoid taking the interphase as it contains DNA. Also be careful to avoid taking phenol, which can inhibit the reverse transcription reaction.
Remove heparin from the RNA sample by lithium chloride precipitation. Add lithium chloride to a final concentration of two molar and incubate the samples overnight at minus 20 degrees Celsius on the following day. Thaw the samples at four degrees Celsius and centrifuge at 20, 000 times.
Gravity for 20 minutes at four degrees Celsius. Carefully discard the supernatant and wash the pellet with 80%ethanol centrifuge. Again, discard the supernatant air dry and resuspend the pellet In 150 microliters of RNAs free water to remove any residual lithium chloride precipitate again with 0.1 volume of three molar sodium acetate pH 5.3 and three volumes of 100%Ethanol incubate at minus 20 degrees Celsius for at least two hours.
Centrifuge at top speed for 20 minutes at four degrees Celsius. Wash the transparent pellet carefully with 80%ethanol and air dry. Finally resuspend the pellet with 25 microliters of RNAs free water and keep the samples at minus 80 degrees Celsius.
The success of this protocol in separating a mitochondria containing fraction from cytosolic components is best tested by performing northern analysis and Western analysis on samples from the different isolation steps. In this example, the samples were from before fractionation or the total fraction, the cytosolic fraction and the mitochondrial fraction. Northern analysis was performed for the following mRNAs, COB an RNA.
That is transcribed inside the mitochondria, A CO one and MRNA that encodes a protein that is imported to the mitochondria. A CT one, an Mr.Nna that encodes the cytosolic actin protein and SEC 61 an M nna that encodes an ER resident protein. The bottom panel is a methylene blue staining of the northern membrane where two prominent bands of RNAs 25 s and 18 s are detected.
Since COB is transcribed inside the mitochondria, its signal should be exclusively in the mitochondria. As was observed in this study. An appearance of COB in the cytosolic fraction will indicate mitochondria lysis during preparation.
ACO one encodes a protein that is imported to the mitochondria and appears mostly in the mitochondrial fraction. A CT one encodes a cytosolic protein and therefore appears mostly in the cytosolic fraction. The presence of SEC 61 in the mitochondrial fraction indicates the presence of ER components in this fraction.
The signals of the RNAs appear in both fractions indicating the presence of ribosomes. Western analysis was performed for the following proteins, POR one A mitochondria, outer membrane protein hx, K one A, cytosolic protein CUE one, and ER protein and RPL 39. A ribosomal protein as expected POR one signal was only detected in the mitochondrial fraction.
If POR one signal were detected in the cytosolic fraction, it would suggest dissociation of mitochondrial components during preparation. Also, as expected hx K one was only detected in the cytosolic fraction. The strong CUE one signal in the mitochondrial fraction indicates that significant amounts of ER related material are co isolated in the mitochondrial fraction.
This may be an outcome of the known physical contacts between the ER and mitochondria. RPL 39 signal appears in both fractions, again, confirming the presence of ribosomes in both fractions, excluding the recovery step from the procedure results in the disappearance of ribosomes from the M fraction, which will affect mRNA association with mitochondria. In addition to verifying the quality of separation between mitochondrial and cytosolic components, it is important to confirm that the RNA or proteins in the samples are intact and there are no severe losses of RNA or proteins during sample preparation.
Intact, RNA and proteins should be detected as distinct bands in the analyses as shown here. For c CCP one mRNA, which encodes a protein that is imported to mitochondria, the appearance of additional shorter bands or smears will indicate degradation. Severe losses will result in a significant difference between the total and the relative amount of signal, which was not observed, taken together.
These results validate this protocol as an effective method for the isolation of mRNAs, ribosomes, and proteins in a single procedure. Following this procedure, genome-wide methods like RNA-Seq or proteomic analysis can be performed in order to identify key and unique features of organal functions. After watching this video, you should have a good understanding of how to isolate RNA that is associated with mitochondria.
