The scope of the presented method is to obtain single and pure glands from fresh plant tissue for molecular analysis. This is accomplished by first harvesting a pitcher from the nepenthe plant. The tissue is then directly fixed onto a micro sampling platform on ice to keep the tissue fresh.
During the micro sampling procedure, glands are isolated in a precise manner and transferred to the PCR tube before performing quantitative PCR analysis on the harvested material. Ultimately, mechanical micro sampling technology is used to obtain single and pure glands from napes plants in order to quickly and micro invasively provide specific tissue for further molecular analysis. Carnivals plants are specialists among the flora because they're able to digest animal protein.
Our knowledge how that works on a molecular level is still limited. A good model to address this question is the picture blend of the genius appendix because it has distinct organs, pictures which produces glands, which contribute to this digestion processes. In appendix.
These glands are located inside the pictures at the bottom. For molecular analysis, it's necessary to isolate these broad and flat structures, having a diameter of about 100 to 200 micrometers directly from fresh tissue. To make it more difficult, the glands are covered by a protective hood.
Having a structure similar to a carport necessitating three dimensional observations during sampling, as well as a tool that can move in three dimensions in a delicate manner. We learned that current micro dissection techniques were not made to solve our problems. A laser, for example, failed because of the high water content of the plant tissue.
Therefore, we decided to use and employ a new mechanical micro dissection technique called aika, Namely for isolating parts of tissues or even single cells of 3D structures. It consists of a stereo or sum microscope where a 3D micro manipulator is integrated in such a way that it can easily reach very flat or very high object. It just moves together with a microscope as a pillar to sample on plants, we often use micro capillaries.
However, in the case of appendix glass, we found that the PTO driven micro forceps work more effective For our procedure. With appendix, the platform has been modified. A little VXY stage supports a to report container filled with ice that is big enough to hold both the tissue sample with its holder as well as the where the gland shall be deposed.
The ice makes sure that the gradation processes within the tissue are slowed down so much that it is possible to recognize the tissue's. Original molecular state, The following procedure has been established from the lower portion of a picture. A piece of about one to two square centimeters is excised and then flattened.
The tissue is then fixed on an object glass by means of double-sided tape. This tissue sample is covered with RNAs free water and is maintained wet during the sampling process. The sample is then positioned with the glands, protective hoods facing the manipulator side.
The angle between sample and tool needs to be adjusted so that the glands are lying flat. Using a universal stage, the tilt can be adjusted in two axes and can be corrected easily under the microscope. The metal stage keeps the sample cool under the microscope.
The glands are well distinguished. Fluorescence helps to easily recognize the contour of both gland and protective hood. The sample is observed through a FSE filter for the isolation of glands.
For RNA extraction, a UV filter is used. Very precise positioning of the micro forceps is essential for the gland isolation. It is recommended to manipulate the tool in the low speed mode.
With the micro forceps, a gland is approached and the arms are placed at its borders by joystick, the micro forceps are closed delicately. The gland is then lifted from the surrounding tissue. After precise isolation, the intact cavity can be seen presenting the underlying vascular tissue.
The isolated gland is then transferred to the PCR tube and deposited into the prepared buffer. To validate this method, the gene expression of atoma leg protein short, TAP, which is secreted into the pitch of fluid, was determined the gene can be amplified directly from the glands without a prior extraction of genomic DNA. Just by adding the PCR reaction mix to the isolated tissue.
The series of experiments involved glands from differently treated nepenthe pictures, as well as samples from their surrounding epidermal tissue. For each experiment total, RNA was extracted. The obtained mRNA was translated into the corresponding CDNA by reverse transcription and used for the subsequent PCR reaction.
To amplify the gene sequence of interest, realtime PCR was used to compare the TLP expression level in nepenthes glands and epidermis. The results show that the TLP transcript level is higher in the secretory glands as compared to the epidermal tissue in both nepenthes allotta and nepenthes miis as visualized by an epidermal tissue over gland tissue ratio of less than one to test whether contact with prey could induce gene expression. TLP transcript levels and untreated pictures and pictures that were fed with drosophila melan gaster were compared after prey trapping.
The epidermal tissue exhibited a higher TLP expression here. The TLP transcript level of glands and epidermal tissue and a treated picture of nepenthes miis was compared. The upregulation of TLP expression in both tissue types indicates that the production of pitcher fluid proteins can indeed be induced by prey.
PCR results suggest that feeding effectively alters TLP expression in both tissue types and can shift the expression ratio between gland and epidermis. We developed an efficient method for harvesting cleanse from FA appendix tissue and isolated a sufficient amount of mRNA to conduct gene expression experiments. With this new method we developed, it's not only possible to address transcriptomics in appendix glands, but in addition to that, we can also address metabolomics as well as proteomics.I.
