Name: expression analysis of mammalian linker-histone subtypes
Uploaded: 2012-03-19T12:00:00
Description: Watch this Scientific Journal Video about Expression Analysis of Mammalian Linker-histone Subtypes at JoVE.com

Dette arbejde er støttet af NIH tilskud GM085261 og Georgien Cancer Coalition Distinguished Scholar Award (til YF).

1. Prøvefremstilling og RNA-ekstraktion <ol><li> Før RNA-ekstraktion, skal alle arbejdsflader og pipetter aftørres med 70% ethanol og behandlet med RNase dekontaminering opløsning, såsom RNase Zap. Denne fremgangsmåde reducerer chancerne for RNase kontaminering og RNA-nedbrydning. Brug handsker for alle procedurer. </li><li> At ekstrahere RNA fra musevæv, dissekere organet af interesse fra aflivet mus, og vask af vævet i iskold phosphatpufret saltvand (PBS: 0,13 M NaCl, 5 mM dibasisk natriumphosphat heptahydra...

<ol>
	<li>Fan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H1+linker+histones+are+essential+for+mouse+development+and+affect+nucleosome+spacing+in+vivo.">H1 linker histones are essential for mouse development and affect nucleosome spacing in vivo.</a> Mol. Cell Biol. 23, 4559-4572 (2003).</li><li>Woodcock, C. L., Skoultchi, A. I., Fan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+linker+histone+in+chromatin+structure+and+function:+H1+stoichiometry+and+nucleosome+repeat+length.">Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length.</a> Chromosome Res. 14, 17-25 (2006).</li><li>Shen, X., Gorovsky, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linker+histone+H1+regulates+specific+gene+expression+but+not+global+transcription+in+vivo.">Linker histone H1 regulates specific gene expression but not global transcription in vivo.</a> Cell. 86, 475-483 (1996).</li><li>Alami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+linker-histone+subtypes+differentially+affect+gene+expression+in+vivo.">Mammalian linker-histone subtypes differentially affect gene expression in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 5920-5925 (2003).</li><li>Happel, N., Doenecke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H1+and+its+isoforms:+contribution+to+chromatin+structure+and+function.">Histone H1 and its isoforms: contribution to chromatin structure and function.</a> Gene. 431, 1-12 (2009).</li><li>Clausell, J., Happel, N., Hale, T. K., Doenecke, D., Beato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H1+subtypes+differentially+modulate+chromatin+condensation+without+preventing+ATP-dependent+remodeling+by+SWI&#47;SNF+or+NURF.">Histone H1 subtypes differentially modulate chromatin condensation without preventing ATP-dependent remodeling by SWI&#47;SNF or NURF.</a> PLoS One. 4, 0007243-0007243 (2009).</li><li>Khadake, J. R., Rao, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-+chromatin-condensing+properties+of+rat+testes+H1a+and+H1t+compared+to+those+of+rat+liver+H1bdec;+H1t+is+a+poor+condenser+of+chromatin.">DNA- chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec; H1t is a poor condenser of chromatin.</a> Biochemistry. 34, 15792-15801 (1995).</li><li>Orrego, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+affinity+of+mammalian+histone+H1+somatic+subtypes+for+DNA+and+chromatin.">Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin.</a> BMC Biol. 5, 22-22 (2007).</li><li>Th'ng, J. P., Sung, R., Ye, M., Hendzel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H1+family+histones+in+the+nucleus.+Control+of+binding+and+localization+by+the+C-terminal+domain.">H1 family histones in the nucleus. Control of binding and localization by the C-terminal domain.</a> J. Biol. Chem. 280, 27809-27814 (2005).</li><li>Wang, Z. F., Sirotkin, A. M., Buchold, G. M., Skoultchi, A. I., Marzluff, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+histone+H1+genes:+gene+organization+and+differential+regulation.">The mouse histone H1 genes: gene organization and differential regulation.</a> J. Mol. Biol. 271, 124-138 (1997).</li><li>Brown, D. T., Sittman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+through+overexpression+and+tagging+of+the+variant+type+of+the+mouse+H1e+and+H1c+genes.">Identification through overexpression and tagging of the variant type of the mouse H1e and H1c genes.</a> J. Biol. Chem. 268, 713-718 (1993).</li><li>Fan, Y., Sirotkin, A., Russell, R. G., Ayala, J., Skoultchi, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+somatic+H1+subtypes+are+dispensable+for+mouse+development+even+in+mice+lacking+the+H1(0)+replacement+subtype.">Individual somatic H1 subtypes are dispensable for mouse development even in mice lacking the H1(0) replacement subtype.</a> Mol. Cell. Biol. 21, 7933-7943 (2001).</li><li>Fan, Y., Skoultchi, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+H1+linker+histone+subtypes+and+their+functions+in+mice.">Genetic analysis of H1 linker histone subtypes and their functions in mice.</a> Methods Enzymol. 377, 85-107 (2004).</li><li>Heid, C. A., Stevens, J., Livak, K. J., Williams, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+quantitative+PCR.">Real time quantitative PCR.</a> Genome Res. 6, 986-994 (1996).</li></ol>

Sættet af assays præsenteret her muliggøre omfattende analyse af ekspressionsniveauerne af mammale linker histon undertyper. Korrekt udformet QRT-PCR assays tilvejebringe meget følsomme og nøjagtige målinger af RNA meddelelser fra alle pattedyr histon H1 gener. Den kritiske del af QRT-PCR assays til linkergrupper histon subtype gener er fremstillingen af ​​cDNA under anvendelse af vilkårlig primer baseret revers transkription. mRNA mest histon gener, herunder de fleste H1 gener, som ikke indeholder en lang po...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> RNase Zap </td><td> Applied Biosystems </td><td> AM9780 </td></tr><tr><td> Trizol Reagens </td><td> Invitrogen </td><td> 15596-018 </td></tr><tr><td> SuperScriptIII </td><td> Invitrogen </td><td> 18080-051 </td></tr><tr><td> Absolut ethanol </td><td> Fisher Scientific </td><td> BP2818-4 </td></tr><tr><td> IQ SYBR Green </td><td> Bio-Rad </td><td> 170-8880 </td></tr><tr><td> RNeasy Mini Kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> Deoxyribonuklease I </td><td> Sigma </td><td> AMP-D1 </td></tr><tr><td> Microseal 96-brønds PCR-plade </td><td> Bio-Rad </td><td> MSP-9605 </td></tr><tr><td> Microseal &#39;B&#39; Lim Seals </td><td> Bio-Rad </td><td> MSB-1001 </td&gt; </tr><tr><td> Saccharose </td><td> Agros Organics </td><td> AC40594 </td></tr><tr><td> Sodium dibasisk heptahydrat (Na 2 HPO 4 · 7H 2 O) </td><td> Fisher Scientific </td><td> BP332 </td></tr><tr><td> Natriumchlorid (NaCl) </td><td> American Bioanalytisk </td><td> AB01915 </td></tr><tr><td> Natriumdihydrogenphosphat heptahydrat (NaH 2 PO 4 · 7H2O) </td><td> Fisher Scientific </td><td> BP-330 </td></tr><tr><td> HEPES </td><td> Fisher Scientific </td><td> BP310 </td></tr><tr><td> Fuldstændig Mini proteinaseinhibitor cocktail tablet </td><td> Roche Applied Science </td><td> 11836153001 </td></tr><tr><td> EDTA </td><td> Sigma </td><td> E-5134 </td></tr><tr><td> Phenylmethansulfonylfluorid (PMSF) </td><td> American Bioanalytisk </td><td> AB01620 </td></tr><tr><td> Nonidet-40 (NP-40) </td&gt; <td> American Bioanalytisk </td><td> AB01425 </td></tr><tr><td> Kaliumchlorid (KCl) </td><td> Fisher Scientific </td><td> BP366 </td></tr><tr><td> Tris [hydroxymethyl aminomethan] </td><td> American Bioanalytisk </td><td> AB02000 </td></tr><tr><td> Magnesiumchlorid (MgCl2) </td><td> Fisher Scientific </td><td> BP214 </td></tr><tr><td> Svovlsyre (H2SO4) </td><td> VWR </td><td> VW3648-3 </td></tr><tr><td> Ammoniumhydroxid (NH4OH) </td><td> Agros Organics </td><td> AC42330 </td></tr><tr><td> Bradford-proteinassay </td><td> Bio-Rad </td><td> 500-0001 </td></tr><tr><td> Acetonitril </td><td> EMD </td><td> AX0145-1 </td></tr><tr><td> Trifluoreddikesyre (TFA) </td><td> JTBaker </td><td> 9470-01 </td></tr></tbody></table>

expression analysis of mammalian linker-histone subtypes

Linkeren histon H1 binder til nukleosom kernepartikel og linker-DNA, der letter foldningen af ​​kromatin i højere orden struktur. H1 er afgørende for pattedyr udvikling 1 og regulerer genekspression in vivo 2-4. Blandt de højkonserverede histon proteiner, er familien af ​​H1 linkersekvenser histoner mest heterogen gruppe. Der er 11 H1 undertyper i pattedyr, der differentielt reguleret under udvikling og i forskellige celletyper. Disse H1 undertyper omfatter 5 somatiske H1S (H1A-e), udskiftning H1 0, 4 kønsceller specifikke H1 undertyper, og H1x 5. Tilstedeværelsen af multiple H1 undertyper, der afviger i DNA bindingsaffinitet og chromatin kompaktering evne 6-9 tilvejebringer et yderligere modulation af kromatin funktion. Således kvantitativt udtryk analyse af de enkelte H1 undertyper, både mRNA og proteiner, er nødvendig for en bedre forståelse af reguleringen af ​​højereFor chromatin struktur og funktion. Her beskriver vi en række assays designet til at analysere ekspressionsniveauerne af individuelle H1 undertype (figur 1). mRNA-ekspression af forskellige H1 variantgener måles ved en række af meget følsomme og kvantitativ revers transkription-PCR (QRT-PCR) assays, der er hurtigere og mere præcis og kræver meget mindre prøver sammenlignet med den alternative fremgangsmåde til Northern blot-analyse. I modsætning til andre cellulære mRNA meddelelser, mangler mRNA&#39;er for de fleste histon gener, herunder størstedelen af H1 gener, en lang polyA hale, men indeholder en stilk-loop-struktur i 3&#39;-utranslaterede region (UTR) 10. Derfor er cDNA&#39;er fremstillet fra totalt RNA ved revers transkription under anvendelse af tilfældige primere i stedet for oligo-dT-primere. Realtid PCR-assays med primere specifikke for hver H1 undertype (tabel 1) udføres for at opnå meget kvantitativ måling af mRNA-niveauer af individuelle H1 undertypesek. Angivelse af rengøring gener analyseres som kontrol for normalisering. Den relative rigelighed af proteiner af hver H1 undertype og kerne histoner opnås ved omvendt fase højtydende væskekromatografi (RP-HPLC)-analyse af totalt histoner ekstraheret fra pattedyrceller 11-13. HPLC-metoden og elueringsbetingelser beskrevet her give optimale separation af muse H1 undertype. Ved kvantificering HPLC profil, beregner vi den relative andel af de individuelle H1 undertype i H1 familien, såvel som bestemmelse af H1 nukleosom forholdet i cellerne.

Watch this Scientific Journal Video about Expression Analysis of Mammalian Linker-histone Subtypes at JoVE.com

Expression Analysis of Mammalian Linker-histone Subtypes

Beskriver vi en række assays til at analysere ekspressionsniveauer af H1 linker histoner. mRNA individuelle H1 gener kvantitativt ved tilfældig primer baseret revers transkription efterfulgt af real-time PCR, hvorimod protein kvantificering af H1 histoner opnås ved HPLC-analyse.

Ekspression Analyse af mammale Linker-histon Undertyper

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Research

JoVE Journal

Biology

Bacterial Gene Expression Analysis Using Microarrays

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

Analyse af Cell Cycle Position i pattedyrceller

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...