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Development of technique for gene mapping of the brain with subcellular resolution

Palo, Sara LU (2019) KBKM05 20192
Pure and Applied Biochemistry
Theoretical Chemistry
Abstract
To develop treatments for currently uncurable neurological disorders, more knowledge is needed of the involved neural circuits and changes in gene expression. The padlock probe technique, in which linear DNA probes are circularized and amplified when hybridized to their cDNA target sequence, has previously been used for both multiplexed gene detection and neuronal projection mapping in brain tissue through in situ sequencing of the resulting rolling circle products (RCPs). cDNA can be synthesized from mRNA captured from tissue onto glass slides, and will remain bound to the glass even after the tissue is removed. By applying padlock probes to this cDNA instead of cDNA crosslinked to tissue, the signal-to-noise ratio can be increased and... (More)
To develop treatments for currently uncurable neurological disorders, more knowledge is needed of the involved neural circuits and changes in gene expression. The padlock probe technique, in which linear DNA probes are circularized and amplified when hybridized to their cDNA target sequence, has previously been used for both multiplexed gene detection and neuronal projection mapping in brain tissue through in situ sequencing of the resulting rolling circle products (RCPs). cDNA can be synthesized from mRNA captured from tissue onto glass slides, and will remain bound to the glass even after the tissue is removed. By applying padlock probes to this cDNA instead of cDNA crosslinked to tissue, the signal-to-noise ratio can be increased and the RCPs kept in the same focal plane instead of spread out through the tissue, in theory allowing for faster imaging.

In this project, the padlock probe technique was applied on mRNA capture slides to further validate and compare this method to the established in-tissue method. It was found that the on-glass method can generate a high RCP density, although the lack of replicates and standardized experiments makes it too early to conclude whether it is overall better than the in-tissue method. A method for automated image processing and quantification was designed and optimized, but found to be lacking when applied to lower quality images. Poor image quality was a reoccurring issue which will hopefully be resolved through changes in imaging equipment and fluorophores. A more thorough analysis of the microscopy images and more standardized experiments in the lab are needed before any final conclusions can be drawn, but initial results are promising enough to motivate further investigation. (Less)
Popular Abstract
The human brain is composed of billions of neurons connected to each other in an intricate network. In order to develop treatments for currently uncurable neurological disorders, more knowledge is needed of how the neurons are connected and how the gene expression in them changes with disease. One way to study these matters is the padlock probe technique, where a DNA molecule binds to a sequence corresponding to a certain gene, and is then closed into a circle and used by a DNA polymerase to create a rolling circle product (RCP). The RCP consists of up to several thousand copies of the same sequence and is thus a strong signal amplifier. The RCPs can be detected by addition of fluorescent DNA molecules which bind to them and show them as... (More)
The human brain is composed of billions of neurons connected to each other in an intricate network. In order to develop treatments for currently uncurable neurological disorders, more knowledge is needed of how the neurons are connected and how the gene expression in them changes with disease. One way to study these matters is the padlock probe technique, where a DNA molecule binds to a sequence corresponding to a certain gene, and is then closed into a circle and used by a DNA polymerase to create a rolling circle product (RCP). The RCP consists of up to several thousand copies of the same sequence and is thus a strong signal amplifier. The RCPs can be detected by addition of fluorescent DNA molecules which bind to them and show them as bright, colorful spots in the microscope, or through a more complex method where the actual DNA sequence of the RCP is determined. Since the RCPs are kept in the same place throughout the entire process, it can be determined which cell in the brain sample the gene in question belongs to.

Padlock probes are usually applied to DNA which has been created from mRNA, the molecule that shows which genes are currently expressed, and the DNA is then in turn bound to its surrounding tissue. However, it is also possible to release mRNA from the tissue and bind it to glass before turning it into cDNA. The tissue can then be removed, and when the padlock probes are applied the RCPs are more clearly distinguishable from the background and thus easier to image.

In this project, the padlock probe technique was applied to DNA bound to glass, and this method was compared to the established one where the DNA is bound to the tissue. It was found that applying padlock probes to DNA bound to glass can result in a high density of RCPs, corresponding to many copies of the gene being expressed in a cell, but there were not enough experiments performed in the same way to determine whether this version of the method is overall better than the established in-tissue version. A reoccurring issue throughout the project was that microscope images were not high enough quality to easily analyze, which also made it more difficult to draw conclusions from the results. However, it has now been established that the new on-glass method can give results that from a first analysis seem to indicate that it is more likely to detect a gene than the in-tissue method. A more thorough analysis of the microscopy images and more standardized experiments in the lab are needed before any final conclusions can be drawn, but initial results are promising enough to motivate further investigation. (Less)
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author
Palo, Sara LU
supervisor
organization
alternative title
Utveckling av teknik för genkartläggning av hjärnan med subcellulär upplösning
course
KBKM05 20192
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8998833
date added to LUP
2020-02-03 13:07:01
date last changed
2020-02-03 13:07:01
@misc{8998833,
  abstract     = {To develop treatments for currently uncurable neurological disorders, more knowledge is needed of the involved neural circuits and changes in gene expression. The padlock probe technique, in which linear DNA probes are circularized and amplified when hybridized to their cDNA target sequence, has previously been used for both multiplexed gene detection and neuronal projection mapping in brain tissue through in situ sequencing of the resulting rolling circle products (RCPs). cDNA can be synthesized from mRNA captured from tissue onto glass slides, and will remain bound to the glass even after the tissue is removed. By applying padlock probes to this cDNA instead of cDNA crosslinked to tissue, the signal-to-noise ratio can be increased and the RCPs kept in the same focal plane instead of spread out through the tissue, in theory allowing for faster imaging. 

In this project, the padlock probe technique was applied on mRNA capture slides to further validate and compare this method to the established in-tissue method. It was found that the on-glass method can generate a high RCP density, although the lack of replicates and standardized experiments makes it too early to conclude whether it is overall better than the in-tissue method. A method for automated image processing and quantification was designed and optimized, but found to be lacking when applied to lower quality images. Poor image quality was a reoccurring issue which will hopefully be resolved through changes in imaging equipment and fluorophores. A more thorough analysis of the microscopy images and more standardized experiments in the lab are needed before any final conclusions can be drawn, but initial results are promising enough to motivate further investigation.},
  author       = {Palo, Sara},
  language     = {eng},
  note         = {Student Paper},
  title        = {Development of technique for gene mapping of the brain with subcellular resolution},
  year         = {2019},
}