Quantitative Gene Regulation

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6 RNA-Seq data normalization?
By Magdy Alabady · University of Georgia

Michael Black · The Hamner Institutes for Health Sciences

In either case, we never know the "real" number of transcripts per gene. With RNA-Seq, that would require that the entire process, from sample collec... [more]

In either case, we never know the "real" number of transcripts per gene. With RNA-Seq, that would require that the entire process, from sample collection to library prep., to sequencing and instrument signal capture was 100% effective. It would also assume we can map those sequences exactly and with complete certainty to their respective positions in the genome and thus count the transcript they land on with complete certainty. Any estimate of relative transcript abundance, be it count data or fluorescence signal intensity is a sampling estimate of what is actually there in the cell/sample. Even when we do summarize reads as counts, we make decisions about what is and is not a valid count. Mapped reads do not all land cleanly within a transcript boundary, so mapping algorithms have to apply cutoffs and decision rules to arrive at an actual discrete count for any given transcript. That inherently means there is uncertainty in the number arrived at, and hence fractional estimates are perfectly valid, as it is an estimate derived from a distribution of hits. Ultimately, it all is estimation and we can never say we know what the actual transcript abundance is for any transcript or gene. All we can do is estimate them from a distribution of counts obtained by sequence sampling.

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9 Can anyone recommend a good protocol for C. elegans spike-in for miRNA from serum?
By Anne (Mulligan Tuttle) Gannon · Health Canada

Anne (Mulligan Tuttle) Gannon · Health Canada

Minakshi, I haven't run my samples yet. I wanted to see if the cel spike in was what most recommended before going ahead, as I don't have a tonne of ... [more]

Minakshi, I haven't run my samples yet. I wanted to see if the cel spike in was what most recommended before going ahead, as I don't have a tonne of sample. I'll let you know how it turns out.

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19 ChIP-Seq analysis for only a number of several defined promoters?
By Maria Thomas · Institut für klinische Pharmakologie

Daniel Cohen · University of Pennsylvania

Maria- if I understand you correctly, you have already identified your TF of interest and feel confident that its binding is dependent on a particular... [more]

Maria- if I understand you correctly, you have already identified your TF of interest and feel confident that its binding is dependent on a particular, known, DNA motif. The problem is that the motif occurs multiple times in the candidate promoters, right? In many cases, factors that strongly induce a target gene will bind multiple sites in the promoter. So in some cases, the prediction of several candidate sites (although probably not exceeding 5-10), is potentially relevant. If you want to find out which of these are function, there are a couple of strategies you could use. 1) genome-wide ChIP-Exo 2) if you are working in mouse or human cells, use the ENCODE datasets at UCSC to look at known DNase HSS sites, and known TF binding sites. TFs tend to bind in groups, so if your motif maps to a known DNAse HSS site or an ENCODE TF cluster, it is more likely to be functional than those that do not. 3) clone some promoters of interest into a luciferase reporter and systematically mutagenize your motifs of interest (although this is not possible for dozens of sites or all your promoters at once). Perhaps #3 is best suited to your system, since it sounds like you are trying to determine whether a particular TF is responding to a stimulus given to your cells. In this case, you might even perform a deletion series in order to map which portion of the promoter responds to your stimulus of interest, and see if the motif is also present in this region, and subsequently whether it is required (effect of a targeted mutation).

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4 RNA-seq data
By Magdy Alabady · University of Georgia

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8 Can I store blood in liquid nitrogen for 2-3 weeks and then isolate the total RNA?
By Arunabha Chakrabarti · Saha Institute of Nuclear Physics

Valentina Guida · Università Vita-Salute San Raffaele

You can store pellet of cells or you can add stabilizing reagent or we also use a special tube "RNA PAX blood " .With this special tubes you can take... [more]

You can store pellet of cells or you can add stabilizing reagent or we also use a special tube "RNA PAX blood " .With this special tubes you can take your blood sample , than keep your blood at 4°C for a short period or store your tube at -80° for years.

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1 How to biologically validate gene interaction for a particular disease?
By Khalid Raza · Jamia Millia Islamia

Eric Jakobsson · University of Illinois, Urbana-Champaign

In addition to gene interaction networks databases (BioGrid, Kegg, String) you might want to try an ab initio tool for measuring similarity among prom... [more]

In addition to gene interaction networks databases (BioGrid, Kegg, String) you might want to try an ab initio tool for measuring similarity among promoter regions called D2Z. Contact Ruth Kantorovitz (Mathematics University of Illinois) for details if you wish to follow up.

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6 RNA seq normalization
By Navonil De Sarkar · Indian Statistical Institute

Navonil De Sarkar · Indian Statistical Institute

Thank you

Thank you

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22 How to identify transcription factors binding to a specific DNA sequence?
By Pankaj Bagul · Indian Institute of Chemical Technology

Diego Ojeda · Universidad Antonio Nariño

Hi, I highly recommend Genomatix software, it is an excellent tool because you can use it for different kinds of analysis such as: NGS, Methylation an... [more]

Hi, I highly recommend Genomatix software, it is an excellent tool because you can use it for different kinds of analysis such as: NGS, Methylation and of course to identify transcription factors in a specific sequence ( MatInspector tool). However Genomatix is not for free, you must pay a 1-year license, but it is a great software. http://www.genomatix.de/

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4 Is there any kit available for especially work for c-DNA synthesis from bacterial m-RNA?
By Ashraf Hussain · University of Science, Malaysia

Alexandro Rodríguez-Rojas · Postdoctoral Researcher Associate

Hi Ashraf. Independently of the kit that you decide to use, what I recommend is to use a reactive from Quiagen, RNAprotect Bacteria Reagent. In bacter... [more]

Hi Ashraf. Independently of the kit that you decide to use, what I recommend is to use a reactive from Quiagen, RNAprotect Bacteria Reagent. In bacteria transcription, translation and almost mRNA degradation are happening simultaneously. This reactive provides immediate stabilization of RNA prior to RNA isolation procedure. It is specially critical with low abundant mRNA. In the past, I had some problems with poorly transcribed RNA and this is the only solution I found. After that, any normal kit with random hexmers should be Ok.

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3 Which is the best online tool for microRNA analysis and target prediction?
By Rakesh Sinha · Agriculture and Agri-Food Canada

Andreas Neueder · King's College London

Hi there, got a couple more links for you (some were already mentioned) http://www.labome.com/about/microRNA-web-resources.html http://www.ebi.ac.uk/e... [more]

Hi there, got a couple more links for you (some were already mentioned) http://www.labome.com/about/microRNA-web-resources.html http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/ http://www.targetscan.org/ http://www.microrna.org/microrna/home.do http://pictar.mdc-berlin.de/ http://mirnamap.mbc.nctu.edu.tw/index.php http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/index.html Best, Andreas

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Answer added to:
31 Troubles with RNA extraction from mouse skin.
By Maria Arismendi Gonçalves · Universidade Federal de São Paulo

Natalia Laspina · University of Buenos Aires

I think everyone agreed you must put your samples in liquid nitrogen just after taken them, and then you can leave them at -80ºC until they are proce... [more]

I think everyone agreed you must put your samples in liquid nitrogen just after taken them, and then you can leave them at -80ºC until they are processed. You must try this in the first place. Next, you must homogeneize the tissue the best you can. this is an important (almost the most important!) step. Then you can go on with your usual protocol. I hope you tell us if something worked for you.

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1 Does anyone have experience with phospholipase C mediated release of GPI anchored proteins on the membrane?
By Sivasai Balivada · Kansas State University

Felix Elortza · Center for Cooperative Research in Biosciences

Hello, Did you use PI-PLC by adding directly to cells? I have no experience on that, but is described elsewhere that it should work (check concnetrati... [more]

Hello, Did you use PI-PLC by adding directly to cells? I have no experience on that, but is described elsewhere that it should work (check concnetrations just in case). You could also try to get membranes, and then perform the two-phase separation by Triton-X114 in combination with PI-PLC. This approach has been proved to work with many GPI-anchored proteins.You could also try with phospholipase D (PLD), although I am not aware of highly purified PLD on marked (I worked with it in a proteomics context, and purity of available ones was not good enough) Good luck! Felix Elortza

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Question:
Open Which are the genes involved in alcohol metabolism?
Genes and Alcoholism

Genes and Alcoholism

By Mariano Rivero Guzman · Instituto Tecnológico y de Estudios Superiores de Monterrey

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24 What is the best approach for statistical analysis of qPCR using the comparative Ct method?
By Diego Mazzotti · Universidade Federal de São Paulo

Deleted

Download and use: https://docs.google.com/file/d/0B15muZJPLjJgZ2VhV3pNRnFweUk/edit?usp=sharing https://docs.google.com/file/d/0B15muZJPLjJgeTQxSVgxNE... [more]

Download and use: https://docs.google.com/file/d/0B15muZJPLjJgZ2VhV3pNRnFweUk/edit?usp=sharing https://docs.google.com/file/d/0B15muZJPLjJgeTQxSVgxNExOR0U/edit?usp=sharing

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Answer added to:
1 What is the biological significance of Higher A, G, C, T count, respectively?
By Sabahuddin Ahmad · Jamia Millia Islamia

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4 What's the best way to store tissues for Chomatin IP?
By Dou Samir · Institut Polytechnique LaSalle Beauvais

Dou Samir · Institut Polytechnique LaSalle Beauvais

Hello, tissues are from animals

Hello, tissues are from animals

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6 What is the most efficient method of storing PBMCs for RNA extraction? Also in how much time should the blood sample be processed for cell separation?
By Iqra Hameed · University of Kashmir

Iqra Hameed · University of Kashmir

Thank you all, will share my experiences with you guys soon. Thanks again

Thank you all, will share my experiences with you guys soon. Thanks again

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Answer added to:
47 How small of a "fold-change" in gene expression can be reliably measured by RT-qPCR?
By David Barker · University of Louisville

Catherine Vaquero · Institut national de la santé et de la recherche médicale

In fact the best thing to do is to look at the level of expression of the corresponding proteins since the level of mRNA does not correlate with the p... [more]

In fact the best thing to do is to look at the level of expression of the corresponding proteins since the level of mRNA does not correlate with the protein translation amplification.

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4 Does anyone know of a resource detailing RNA expression in wild type HEK 293 cells?
By Kristopher Bosse · The Children's Hospital of Philadelphia

Thomas Albert · Westfälische Wilhelms-Universität Münster

Hi Kristopher, there are currently 1333 hits in GEO DataSets if you search for HEK293; numerous contain "wildtype" 293's, e.g. series GSE1676... And I... [more]

Hi Kristopher, there are currently 1333 hits in GEO DataSets if you search for HEK293; numerous contain "wildtype" 293's, e.g. series GSE1676... And I agree with Maximilian that it is probably a good idea to look from the Tx factor direction into these GSE sets, otherwise you might get lost in the wealth of data...

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Question:
Open Can anyone give me advice for working with the Ribotag (BAC TRAP) system related isolation of mRNA?
mRNAs associated with HA/GFP tagged ribosomes are pulled down with HA/GFP antibodies coupled to magnetic beads and then isolated and analyzed by micro... [more]

mRNAs associated with HA/GFP tagged ribosomes are pulled down with HA/GFP antibodies coupled to magnetic beads and then isolated and analyzed by microarray.

By Joshua Ortiz-Guzman · Baylor College of Medicine

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Answer added to:
6 Why is the elongation step generally not performed in real-time PCR ?
By Béatrice Gaume · Université des Antilles et de la Guyane

Siva Uppalapati · Defence Research and Development Organisation

hi, Polymerases from different manufacturers or from different sources have different temparatures for their optimal activity. In the current case, th... [more]

hi, Polymerases from different manufacturers or from different sources have different temparatures for their optimal activity. In the current case, the polymerase is from Sulfolobus solfataricus. Kindly read this article for better understanding of how this enzyme is designed... A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro by Wang et al., 2004. The optimal activity of this enzyme should be at 60 C.. hence there would be no need for an extra extension time.

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Question:
Open Difference between normalized and relative luciferase activity?
Am I right if I say that when luciferase activity is normalized using untreated Firefly activity, it is called Normalized Luciferase Activity and when... [more]

Am I right if I say that when luciferase activity is normalized using untreated Firefly activity, it is called Normalized Luciferase Activity and when normalized using Renilla luciferase activity, it is called Relative Luciferase activity?

By Paritosh Parashar · Indian Institute of Technology Kanpur

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Question:
Open Has anyone used a protocol for microplate assay, for E. coli cells with fluoresce in di-ß-D-galactopyranoside (FDG) substrate, but with intact cells?
I want to measure β-galactosidase activity of a promoter fused to lacZ gene as reporter. The protocols that I have found using cell lysates or measur... [more]

I want to measure β-galactosidase activity of a promoter fused to lacZ gene as reporter. The protocols that I have found using cell lysates or measured activity in short time ranges. I want to do a kinetics.

By Victoria Grosso · National Autonomous University of Mexico

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Answer added to:
2 Does anybody have rest 2009 software?
By Suriya Ramiah · Putra University, Malaysia

Brian Lin · Tufts University

It is available here: http://b2b.qiagen.com/products/rest2009software.aspx#Tabs=t2 with free login to qiagen. Has anyone gotten this to work on window... [more]

It is available here: http://b2b.qiagen.com/products/rest2009software.aspx#Tabs=t2 with free login to qiagen. Has anyone gotten this to work on windows 7? I gave up a while ago, but never asked it here

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2 How to design/choose an effective primer for RT-qPCR?
By Oskan Tasinov · Medical University of Varna

Oskan Tasinov · Medical University of Varna

I'm using cDNA to perform Real Time - qPCR. I work on gene expression of antioxidant and inflammatory related enzymes. Thank you for suggestion!

I'm using cDNA to perform Real Time - qPCR. I work on gene expression of antioxidant and inflammatory related enzymes. Thank you for suggestion!

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About Quantitative Gene Regulation

A group for scientists interested in quantitative descriptions of gene regulation in pro- and eukaryotes (equilibrium and non-equilibrium protein binding, chromatin rearrangements, covalent modifications, input-output cis-regulatory functions, etc) using approaches of biophysics, molecular and cell biology, bioinformatics, systems biology and synthetic biology.

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Grace Lachica

University of the Philippines Los Baños
Prathyusha Konda

Indian Institute of Technology Madras
Mohammad Sadeq Alizadeh

Institute for Advanced Studies in Basic Sciences
Sandrine Morlot

Institut de Génétique et de Biologie Moléculaire et Cellulaire
Jean-Pierre Aimé

Université Bordeaux 1
Ramos Duran

Infanta Luisa Hospital
Rezvan Shahriary

Islamic Azad University of Najafabad
Katharina Schiessl

John Innes Centre
Lizhong Liu

The University of Hong Kong
Mahmoud Abu Moustafa

Central University of Finance and Economics

Quantitative Gene Regulation

About Quantitative Gene Regulation

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