The following was produced by researchers at the University of Georgia and the Warnell School of Forestry and Natural Resources. This demonstration video describes the harvesting of xylem and phem tissue and subsequent RNA isolation from Pinus Teta, commonly known as lob lolly pine, which is the single most important conifer species for forest products production in the United States. Once the tree is failed, in this case, a 40-year-old tree, the main stem is cut into bolts that are approximately 50 centimeters in length.
The bolt is then scored longitudinally with the chainsaw in several places to facilitate removal of the bark. Using a hammer and wood chisel, the bark is litted up until it can be peeled away in sections. Next, the flowing tissue is removed by hand and is stripped from the inside of the bark sections and immediately immersed into liquid nitrogen.
Once the bark has been removed, the thin layer of secondary xylem can be collected by scraping the bolt surface with a single-sided razor blade. The watery xylem tissue is immediately placed into liquid nitrogen. In this harvest, we also collected samples of reaction wood, commonly referred to as compression and opposite wood before cutting limbs from the main stem.
The limb orientation is first marked with spray paint so that the areas containing the two tissue types opposite wood on top of the limb and compression wood on the bottom can be determined. During bark removal. The limbs are first scored with a chainsaw along lines delineating compression and opposite wood and bark removal is performed as with the main stem bolts, except that only bark in the region corresponding to the type of reaction.
Wood being harvested is removed at any given time. It is typically more difficult to remove large bark sections from these smaller limbs. Secondary xylem reaction wood samples are collected in the same manner as with normal secondary xylem by scraping with the razor blade and immediate immersion into liquid nitrogen.
Note the reddish brown color characteristic of compression wood. It is noteworthy to mention that collecting and processing multiple samples from a large tree requires many hands with each person involved performing a designated task so that all tissue types are being collected simultaneously to help reduce RNA degradation samples frozen and liquid nitrogen are subsequently removed to labeled storage bags and placed on dry ice for transport back to the laboratory. Our first step is to pulverize the tissue using a specs model 68 50 freezer mill compared to grinding with a mortar and pestle, the step significantly increases RNA extraction yields for woody tissue samples, keeping the samples cold with liquid nitrogen.
The tissue is first broken into smaller pieces by hand and placed into the sample chamber. The chamber is inserted into the carrier locked in place, and the sample is milled for two minutes. The final product has the consistency of a coarse powder.
The RNA extraction protocol is a two day process. On the first day, 2.5 grams of frozen tissue is added to RNA extraction buffer that has been heated to 65 degrees Celsius. The sample is shaken vigorously to mix, and an equal volume of chloroform is added and the sample is mixed again with gentle inversion.
The sample is next placed on a rotating wheel for five minutes. The sample is then transferred to high speed Teflon Oakridge tubes and centrifuge in an SS 34 rotor at 12, 000 RPM for five minutes. The upper aqueous fraction is extracted into a fresh tube and a half volume of chloroform is added.
The sample is vortexed for one minute and again placed on the rotating wheel for five minutes. After a second centrifugation, the aqueous fraction is again extracted into a 50 mil high speed polypropylene tube and centrifuged at 10, 000 RPM for 20 minutes. To remove any residual particulates, the sample is decanted into a 50 mil Falcon tube and a one-quarter volume of 10 molar lithium chloride is added to the sample.
The sample is mixed by shaking and placed at four degrees Celsius overnight. On the second day. The lithium chloride precipitated sample is first centrifuged at 11, 000 RPM for 30 minutes at four degrees Celsius.
The sate is poured off and discarded, and the tubes are inverted onto chem wipes for one minute. To remove any excess liquid, the RNA pellet, which should be visible is resuspended in 800 microliters of hot SSTE buffer that has been preheated to 65 degrees Celsius. Be sure to pipette up and down several times and all around the pellet margins to ensure that it all gets into solution.
Next, transfer the resuspended RNA into a two mil and beyond RNA free micro fuge tube. Heat the sample for one to two minutes in a 65 degree Celsius water bath, followed by vortexing to ensure the sample is completely resuspended. This can be repeated if necessary.
Now, add an equal volume of buffered phenol chloroform. Vortex the sample for 15 to 20 seconds and micro fuge at 14, 000 RPM for three minutes. The aqueous fraction is extracted into a new micro fuse tube and an equal volume of is added.
The sample is vortex and micro fused as in the previous extraction. For the final organic extraction phase. Lock gel tubes are first micro fued at 11, 000 RPM for two minutes.
Next, the RNA sample from the previous extraction is added to the phase lock gel, followed by a half volume of chloroform, and the two are mixed by pipetting gently. The sample is then micro fued at 11, 000 RPM for five minutes, and the aqueous RNA sample is transferred to a new ambion RNA free micro fuge tube. The next step is a simple RNA precipitation to the sample at a one 10th volume of three molar sodium acetate pH 4.8 and two volumes of 100%ethanol.
The sample is vortex, briefly placed in a minus 80 Celsius freezer for 30 minutes, and then micro fuge for 30 minutes at four degrees Celsius. The supernatant is carefully aspirated and the small white pellet is washed with one mil of ice cold, 70%ethanol aspirate and repeat the ethanol wash after the final aspiration. Residual ethanol is allowed to evaporate by leaving the tubes open on the bench for five minutes.
RNA pellets are resuspended in 100 microliters of DEPC water and resuspended by vortexing and heating at 65 degrees. This can be repeated if necessary. Sample concentration is determined.
Spectro photometrically and 2 62 80 and 2 62 30 ratios are calculated to estimate the extent of protein and carbohydrate contamination in the sample. 1%AGROS gel electrophoresis is used to qualitatively estimate sample quality genomic DNA contamination and RNA degradation Sample integrity of critical samples is evaluated further using an Agilent 2100 bioanalyzer to determine ribosomal 28 18 s ratios and an RNA integrity number. Thank you for watching our video on the harvesting of tissue samples and the isolation of total RNA from LOB lolly pine.
The following individuals are responsible for the filming and production of this training video.
