With a special thanks to the webinar speakers Professor Mathias Uhlén, Ph.D. and Evelina Sjöstedt, Ph.D. from the KTH Royal Institute of Technology Stockholm, Sweden and Jackie Oberst, Ph.D. our moderator from Science/AAAS, for their participation in this webinar sponsored by us at Atlas Antibodies.
Below you will find the follow-up questions with their respective answers provided by Dr. Evelina Sjöstedt together with Dr. Laura Pozzi from Atlas Antibodies.
Q&A on Research Insights
A: The Brain Atlas explores the protein expression in the mammalian brain by visualization and integration of data from three mammalian species (human, pig and mouse).
It is based on an innovative way to look at the brain:
1) a brain-centric perspective comparing the brain regions to each other. Here you can explore which proteins are expressed in a particular part of the brain or where in the brain your protein of interest is expressed and,
2) a whole-body tissue type perspective that considers the brain as a whole tissue. Here you can explore which proteins are expressed in the brain compared to other tissue types in the human body.
A: There are numerous pages where interesting findings, great immunohistochemical examples and tissue/region/organelle-specific features are summarized.
On the main page for the Brain Atlas, you can find entries to summary pages for each of the ten brain regions and the regional classification of the expression data. Make sure to click on the icons to explore the summary pages. From there you can access the individual gene pages classified in that region.
You can find similar classifications on the Tissue Atlas main page.
The Human Protein Atlas website includes a dictionary to facilitate the interpretation and use of the image-based data, but also to serve as a tool for training and understanding tissue histology, pathology and cell biology.
A: The regional classification of Brain Atlas from the HPA is based on available FANTOM5 and GTEx expression data sets and includes regional classification of 17,229 protein-coding genes. All databases use their gene model (ensemble version, etc.) thus leading to a number of genes without data.
A: Yes, we are planning to continue developing the Brain Atlas, implementing relevant data and adding more regional information. We will add more regional information about the cerebral cortex as well.
A: Unfortunately, CSF is not included yet, but it should be added in the future.
A: We are not aware of such a map at the moment, but this is definitely an interesting aim for the future: adding data from brain samples obtained from patient with neurological disorders into the Brain Atlas for an interesting comparison on a large scale.
Q&A on Antibodies & Validation
A: The ‘genetic strategy’ for antibody validation by means of CRISPR/Cas9 system gene-knockout (KO) or by siRNA gene-knockdown (KD) in a suitable cell line, has emerged as an ideal tool for antibody-specificity validation.
The specificity of an antibody staining is evaluated through the analyses of the corresponding target gene from cell lysates samples before and after KO or KD. The antibody is validated when the protein levels correspond to the decreased RNA levels.
The genetic validation method applied for the antibodies used in the Human Protein Atlas project is knockdown by siRNA. Learn more about the genetic validation method here.
A: Due to the origin of the human samples (i.e. biobank) it is not possible to optimize the fixation steps before the antibodies staining. However, the antibody dilution is verified during the optimization of the immunohistochemical protocol.
All antibodies are rigorously validated for specificity and reproducibility and are characterized in several applications. In IHC, each antibody is evaluated in 44 normal tissues and the 20 most common cancerous tissues.
As an additional layer, enhanced validation is performed using several different strategies such as the independent antibodies validation (paired antibodies targeting different epitopes of the same protein are compared) and the orthogonal antibodies validation (the antibody staining is compared to RNA expression levels in corresponding tissues).
A: Only antibodies that pass the minimum criteria of standard antibody validation are published on the Human Protein Atlas website. In addition to the standard quality assurance, enhanced antibody validation strategies are performed in an application-specific manner. Moreover, in order to provide an overview of protein expression patterns, all images of tissues stained by immunohistochemistry are manually annotated by a specialist followed by verification by a second specialist.
A: You can find the recommended protocols for the antibodies developed and used within the Human Protein Atlas project here on the Atlas Antibodies' protocols page.
The antibodies developed and used within the Human Protein Atlas and Brain Atlas are manufactured and distributed by us at Atlas Antibodies under the brand name Triple A PolyclonalsTM.
If you find an antibody you are interested in when browsing the Brain Atlas, click the link "Antibodies and Validation" on the left side navigation. On the following page, you will find the antibodies listed together with a supplier link to the respective product page on Atlas Antibodies website.
You can also find all antibodies here.
Q&A on Technology and Data
A: A major benefit with transcriptomics data is that it is measurable (i.e. quantitative).
In addition, transcriptomics data covers a wide spectrum of genes and a large dynamic range- low abundant/high abundant-gene expression is detected.
Mass spectrometry (MS) could also be quantitative, especially after the introduction of the new QPrEST standards for protein quantification. MS has some limitations not being able to provide data from a large range of gene expression levels.
Yes, in the Human Protein Atlas website there is a section under the menu tab about downloadable data.
The data files include data available in the Human Protein Atlas version 19. A subset of this data can also be downloaded from the Search page with the genes corresponding to the current search result in the result in different formats; XML, RDF, TSV & JSON.
If you have further questions, please contact the Human Protein Atlas.
If you want to use the data for commercial purpose please see the licence & citation page on the Human Protein Atlas website for full details.
A: At the moment, all data is based on ‘normal’ (healthy) tissue and no human neurological disease data is included. The Brain Atlas represents the foundation to understand how the mammalian brain is built. The ability to compare the gene expression in the brains of different mammalian species will serve to validate or select a model system for human disease in the future.
The ages of the human brains are listed together in the sample description for each protein. As an example, here is the sample description for the protein GFAP. The other brain samples were taken from 1-year-old pigs and 2-months-old mice.
This is an interesting and crucial question. Transcriptomics data combined with affinity-based protein in situ localization down to single-cell detail is available on the Brain Atlas as a brain-centric sub-atlas of the Human Protein Atlas. Each gene is provided with a summary page, showing available expression data (mRNA) for summarized regions of the brain as well as protein location for selected targets.
Enriched is defined as 4-fold higher expression in one tissue compared to the whole. Group-enriched is 4-fold higher expression in a group of 2-5 tissues. Enhanced is 4-fold higher expression in one more tissue compared to the MEAN of all other tissues. Please refer to the Human Protein Atlas website, for more details.
In the Brain Atlas, the mRNA expression is based on transcriptomics reads and quantitative numbers, so we do not have the protein location other than information about the tissue/regional origin of the sample.
On the other hand, with the immunohistochemical staining we have detailed information about the protein’s location but less quantitative information about them. The summary bars for regional expression and protein location in the brain is always available.
Moreover, at least for the mouse brain, you can compare the in-situ hybridization (ISH) images of the Allen Brain Atlas with the protein profiling in that we provide in the Brain Atlas.
A: Yes, both male and female genders are represented in our human, pig and mouse brain samples.
A: There are definitely several BBB-related proteins included in the Brain Atlas. We have for instance seen several proteins located to the astrocytic end feet.
A: Most of the human brain samples are from postmortem tissues. A few samples included are surgically removed from living brain.
A: At the moment there is no plan to separate the two. However, it might be possible to activate an ON/OFF selection feature for the two genders (similar to the Pathology Atlas).
A: Yes, we have. We are performing volume immunostaining (iDISCO), on whole mouse brain and on smaller pieces of human tissue samples. As an example, you, watch this video about the 3D distribution of orexin immunostaining in the mouse lateral hypothalamus.
A: Enrichment in tissues or regions, refers to RNA expression levels, whereas cell-type enrichment refers to proteins.
Thanks to the gene classification based on enrichment interesting protein targets were found to further investigate further the proteins and their location.
A: The normalized expression (NX) is used to compare genes expression between tissues, while the protein-transcripts per million (pTPM) is used to compare between genes in the same tissue.
The Human Protein Altas
The Human Protein Atlas is an open-source database divided into six parts, the Tissue Atlas, Cell Atlas, Pathology Atlas, Brain Atlas, Blood Atlas, and Metabolic Atlas.
The Human Protein Atlas project was initiated in 2003 by Swedish researchers, headed by Professor Mathias Uhlén, and funded by the Knut and Alice Wallenberg Foundation to map the human proteome.
Triple A Polyclonals - the antibodies used in the Brain Atlas
The antibodies developed and used in the Brain Atlas are manufactured and made available to fellow researchers by Atlas Antibodies as Triple A PolyclonalsTM. You can search for them using the product numbers starting with "HPA" listed in the Human Protein Atlas.