The overall goal of the following experiment is to determine relative changes in TRNA charging levels. This is achieved by isolating RNA at a low pH before and after treatment to isolate TRNA with intact charging levels. As a second step, the RNA is subjected to a per iodate oxidation treatment, which allows for determination of relative charging levels.
Next ligate samples to a fluorescent oligonucleotide and hybridize on a microarray in order to measure changes in ligation levels due to A-T-R-N-A charging results are obtained. The CHO genomic level changes in TRNA charging based on TRNA microarray technology. Hi, I'm John Zai from the laboratory of DRT Pan in the department of Biochemistry and molecular biology at the University of Chicago.
Today I'll be showing you a procedure for isolating and measuring charge TRNA at the genomic level. We use this to measure changes in TNA charging during stressor puns in our lab. So let's get started.
To isolate total charge to TRNA, start by growing a minimum of the equivalent of 30 OD 600 of yeast cells, which should produce 30 micrograms of total RNA pellet the cells in a tabletop centrifuge. Resuspend cells in a lysis buffer solution. Add an equal volume of sodium acetate, saturated phenyl chloroform and vortex for 30 seconds, followed by icing for at least 30 seconds.
Repeat vortexing and dicing two more times. Centrifuge the sample at 18, 600 RFC for 15 minutes at four degrees Celsius. Then remove the aqueous layer and perform another acetate saturated phenyl chloroform extraction after the second extraction.
Precipitate the RNA by adding 2.7 x volumes of ethanol and centrifusion at 18, 600 RFC for 30 minutes at four degrees Celsius. Resus suspend RNA pellets in lysis buffer solution and then precipitate with ethanol a second time. Finally, re suspend the RNA in a solution containing 10 millimolar buffered acetate and one millimolar EDTA for subsequent treatment.
The sample can be stably stored at minus 80 degrees Celsius for about two weeks longer. Storage is not recommended as some charged TNA will start to deci isolate prior to labeling with S3 or SCI five fluorescent oligonucleotide tags. The total TRNA is subjected to a pariah day oxidation treatment, which allows for determination of relative charging levels mixed the total RNA with 0.066 micromolar of each eco irna standard and 100 millimolar buffered acetate in the presence of 50 millimolar sodium par IID date for the control TRNA sample that will not be oxidized use 50 millimolar sodium chloride in place of the sodium par IID date.
Incubate the reactions for 30 minutes at room temperature after 30 minutes, quench the reaction by adding glucose to 100 millimolar and incubating at room temperature for five minutes. Once the reaction is quenched, perform a buffer exchange using a G 25 spin column to remove any remaining sodium prorate from the samples. Precipitate the samples by adding buffered acetate to a final concentration of 133 millimolar sodium chloride to a final concentration of 66 millimolar and 2.7 x volumes of ethanol.
Next deci, isolate the tRNA samples by Resus, suspending them in 50 millimolar tris HCL and incubating at 37 degrees Celsius for 30 minutes. After 30 minutes, neutralize the reaction by adding an equal volume of 50 millimolar buffered acetate and 100 millimolar sodium chloride precipitate with 2.7 x volumes of ethanol. Re suspend the RNA pellet in water at about one microgram per microliter.
Once the control and oxidized samples have been checked for RNA quality by running on agarose gels, we can proceed with the fluorescent labeling to attach fluorescent oligo tags onto the TRNA. Combine 0.1 microgram per microliter of de RNA with four micromolar SI three or SI five containing oligonucleotides ligase buffer 15%DMSO 0.5 units per microliter of T four DNA ligase and yeast exo phosphatase incubate at 16 degrees Celsius for over 16 hours After ligation, mix the samples with four volumes of potassium acetate and potassium chloride, and then extract with an equal volume of phenyl chloroform following extraction of the aqueous phase precipitate the RNA preparations with ethanol. Finally, resus, suspend the RNA pellets in water to approximately 0.1 microgram per microliter prior to hybridization oil, microarray slides and distilled water for one to two minutes to remove unbound oligonucleotides.
Next, combine the S3 or SCI five labeled samples with 140 microliters of hybridization buffer salmon sperm DNA to a final concentration of 140 micrograms per milliliter and poly A to a final concentration of seven D micrograms per milliliter run a hybridization program on an automatic array hybridizer. We highly recommend the use of an automatic hybridization machine for TRNA work since manual hybridization produces high background. When the hybridization program is complete, manually wash the slides with wash three solution by gently shaking in a 50 milliliter conical tube for three to five minutes.
Wash the slides at least twice in tubes pre-wrapped with aluminum foil. Dry the slides by centrifuging at a low speed for five to 10 minutes for optimal results. The slides should be scanned immediately after they're dried.
Slides can be scanned two to three times without noticeable degradation in image quality representative results of a properly isolated total RNA sample and its analysis are shown here. This sample has a very clean spectrum with an OD two 60 to OD two 80 ratio near two with no detectable protein or phenol contamination when run on 1%agro gels. Both oxidized and control samples should show a strong TNA band with other possible nucleic acid bands varying between organisms.
If a sample is noticeably weaker than other samples, or if smearing is observed, samples should not be used for microarrays. Microarrays should have a very low background, which is an order of magnitude lower than the weakest TRNA probe. I've just shown you how to isolate and determine the relative charging levels of TRNA when doing this procedure.
It's important to remember to keep your reagents free of nucleases, especially during the oxidation and dilation steps. Thanks for watching and good luck with your experiments.
