The overall goal of this procedure is to establish and optimize metabolite and RNA sequencing data from tobacco tree leaf material for systems biology oriented estimations. This is accomplished by first collecting the biological material of a developmental leaf series. The second step is to perform extraction of the material using the appropriate solvents dependent on the subsequent analysis.
Next, metabolites and hydrocarbons are measured using gas chromatography, mass spectrometry or GCMS and the transcripts are analyzed using RNA sequencing data acquisition. Ultimately, the combination of metabolite profiling and transcriptional profiling via RNA sequencing is used to show how tobacco tree leaves develop long chain hydrocarbons and other important compounds. This method can be used to identify and quantify important useful metabolites made in the leaves of the tobacco tree.
Though this method can provide insight into the tobacco tree metabolome. It can also be used for other systems such as tobacco or related species. Generally, individuals new to that method will struggle because tobacco tree leaves are tough to extract good RNA from.
In this multi Bioo PRO video, you will see members from the FERNA group extract and profile metabolize. Using GCMS, you will also see a member from the Fraser Group extract hydrocarbons and also one member from my own group who will extract RNA for RNA sequencing Begin by growing neko Oceana Glauca plants in 30 centimeter diameter pots containing M two professional growing medium as detailed in the text protocol to prepare the primary metabolites, harvest the leaves and stems of the plant and freeze the material immediately lyophilize the frozen plant materials by freeze drying for three days, then grind the lyophilized samples by mixer mill with metal balls, aliquot the resulting fine ground dry materials into a two milliliter centrifuge tube or glass vial. Next aliquot 10 milligrams of leaf or 20 milligrams of stem ground dry plant material into two milliliters screw cap round bottom tubes.
Add 1.4 milliliters of 100%methanol and vortex for 10 seconds. Then add 60 microliters of rib atol as an internal quantitative standard before vortexing. For an additional 10 seconds, add one stainless steel ball and homogenize the material with a mixer mill for two minutes at 25 hertz following centrifugation for 10 minutes.
At 11, 000 times gravity, transfer the supernatant to a glass vial. Add 750 microliters of chloroform and 1.5 milliliters of water before vortexing the mix for 10 seconds. Then transfer 150 microliters from the upper phase into a fresh 1.5 milliliter centrifuge tube.
Next, dry the samples using a speed vac. It is imperative that the samples are reduced to dryness for between three and 24 hours until no residual liquid is present. Once dry, add 40 microliters of methin hydrochloride.
Then shake the mix in a horizontal heat block shaker for two hours at 37 degrees Celsius. Add 70 microliters of M-S-T-F-A mix before shaking again for two hours at 37 degrees Celsius. Spin down the drops on the cover by a short centrifugation before, before transferring the liquid into glass vials suitable for GC MS analysis.
Following GCMS analyze data by configuring software for the analysis of mass spectrometry data and selecting the data analysis to be processed. Prepare a table of detected peaks of interest in accordance with compound class. Identify the peaks by coalition of standard compounds, subsequently annotate detected peaks using mass spectrometry analysis, structural characterization algorithms, literature survey and metabolite database search for hydrocarbon extraction.
Submerge approximately 200 milligrams of freshly harvested ncoa glauco leaves insolvent for two to 20 minutes, remove the leaves and dry off solvents completely by rotary evaporation. Then resuspend the samples in one milliliter of HPLC grade hexane. Proceed to perform GCM s analysis of the samples using the parameters that are detailed in the text protocol.
Initially process the chromatogram components using an automated MS.Deconvolution and identification system and the NIST 98 MS.Library. Confirm the identification of saturated long chain acan hydrocarbons by comparing retention times and classical fragmentation patterns to known authentic standards. Next, determine hydrocarbons quantitatively by comparing integrated peak areas with dose response curves.
Constructed from authentic standards, calculate means and standard error of the means using Excel software. This protocol combines TRIOL RNA extraction with an RN easy kit to obtain high quality RNA grind 100 milligrams of frozen plant material in two milliliter centrifuge tubes with a metal ball by a mixer mill. Following sample homogenization, add one milliliter of triol after centrifuging the material for 10 minutes at 12, 000 times gravity and four degrees Celsius.
Transfer the supernatant into a new reaction tube. Add 200 microliters of chloroform and invert the tube several times before incubating for three minutes at room temperature. After centrifuging the mix for 15 minutes.
As before, transfer the upper aqueous phase into a new reaction tube. Add approximately 2.5 volumes of ethanol. Invert the tube several times and incubate for 30 minutes at minus 80 degrees celsius.
Next, move the sample including any precipitate to a spin column. Centrifuge the column for 15 seconds at 8, 000 times gravity and discard the flow through. Add 700 microliters of wash buffer RW one, which is provided in the extraction kit to the spin column and centrifuge.
Again, after discarding the flow through, add 500 microliters of the second wash buffer, RPE also provided by the kit to the spin column. Centrifuge the column again and discard the flow through before adding more buffer RPE and centrifuging. As before, then place the spin column into a new 1.5 milliliter reaction tube and add 50 microliters of RNAs free water centrifuge, the column for one minute at 8, 000 times gravity to elute, the RNA.
Remove the residual DNA using a DNA free kit according to manufacturer's instructions. As a final step, determine the quality of the RNA. The RNA should have RNA integrity numbers above eight before sending off the RNA for next generation sequencing.
The HPLC profile shows a representative result of the isoprenoid analysis of ncoa glauca leaf extracts. The different ISOPRENOIDS of C 40 and above were detected using a photo diode array detector. The peaks were annotated based on code chromatography and spectral comparison between authentic standards.
The two MS chromatograms are the result of primary metabolite analysis from ncoa, glauco leaf and stem material. The MS spectrum of a peak corresponding to serine is also given, as an example, representative output of the bioanalyzer used for determining the quality of the RNA is shown. The two main peaks in the chromatogram correspond to 18 s and 25 s ribosomal, RNA indicating intact RNA in this sample.
Additional peaks of fragmented ribosomal RNA would appear in case of partially or heavily degraded RNA Once mastered. This technique can be performed rapidly and it is compatible with the analysis of other metabolites Following this procedure. Other methods like realtime PCR or other ways of analyzing RNA can be used in order to answer additional questions like production of small RNAs or expression of specific genes.
After watching this video, you should have very good understanding on how to extract and evaluate metabolite data from the tobacco tree.
