Molecular Cloning, also known as Maniatis, has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential.
TY - CHAP. T1 - 5′-Monopyrene and 5′-Bispyrene 2′-O-methyl RNA Probes for Detection of RNA Mismatches. AU - Novopashina, D. S.. AU - Semikolenova, O. A.. AU - Venyaminova, A. G.. PY - 2020/1/1. Y1 - 2020/1/1. N2 - Progress in synthesis of novel fluorescent oligonucleotides has provided effective instruments for nucleic acid detection. Pyrene conjugated oligonucleotides have demonstrated their effectiveness as fluorescent hybridization probes. Here we describe the synthesis, isolation, and analysis of 5′-monopyrene and 5′-bispyrene conjugates of oligo(2′-O-methylribonucleotides) and their application as probes for fluorescent detection of mismatches in RNA targets.. AB - Progress in synthesis of novel fluorescent oligonucleotides has provided effective instruments for nucleic acid detection. Pyrene conjugated oligonucleotides have demonstrated their effectiveness as fluorescent hybridization probes. Here we describe the synthesis, isolation, and analysis of 5′-monopyrene and ...
Embryos. Pregnant CD-1 mice (Charles River Laboratories, Wilmington, MA) were killed, and the litters were collected according to the NIH Guide for the Care and Use of Laboratory Animals protocol. Embryos were individually staged according to the method of Theiler (1989).. Probes. The in situ hybridization probe forFng was obtained by screening an 8.5 d postcoitum (dpc) embryonic mouse λgt10 library (a gift from Brigid Hogan) with a mixture of human (Expressed sequence tags, Research Genetics) and chicken Fng (Laufer et al., 1997) cDNAs. Several strongly hybridizing clones were obtained, and one of these, pMFR1, was selected for further analysis. The 2.2 kb insert of pMFR1 contains the entire coding region of murine Fng (Cohen et al., 1997; Johnston et al., 1997). The antisense RNA probe was generated by using T3 RNA polymerase after HindIII restriction digest of pMFR1, and the sense RNA probe was generated by T7 RNA polymerase afterXbaI restriction digest. The NT-3 RNA probe was generated from ...
Parts of the cDNA fragments were ligated into pBluescript II KS vector, and we used this plasmid to synthesize a Dig (digoxygenin)-labeled RNA probe. Dig-labeled RNA probes were made by in vitro transcription from the linearized plasmid with a Dig RNA labeling kit (Roche). T7 or T3 RNA polymerase (Roche) was used in the in vitro transcription to synthesize an antisense RNA probe for hybridization.. In each step, it took 15 min to form a drop of dicyemids in the bottom of each microtube. Dicyemids were washed 5 times and then fixed overnight at 4°C in 4% paraformaldehyde in 0.5 M NaCl and 0.1 M MOPS buffer. After fixation, the dicyemids were dehydrated in an ethanol series (30%, 50%, 70%) for 10 min each and the dicyemids were stored in a 70% ethanol up-series at -30°C. Dicyemids were hydrated in an ethanol down-series (70%, 50%, 30%) for 5 min each, and were washed 3 times in PBT (phosphate buffer saline [PBS] with 0.1% Tween 20). The hydrated dicyemids were partly digested by protease K (0.5 ...
mutaFISH™ EGFR T790M T790wt RNA Probes is designed to detect human EGFR T790M gene mutation on single strand RNA in cells using in situ rolling-circle amplication technology. (FP0001) - Products - Abnova
Fig. 6. Spatial expression profile of enpp1, 4, 6 and 7 during development. Whole mount in situ hybridisation with DIG-labelled antisense RNA probes was performed on embryos from stages 5-41 for enpp1, 4, 6 and stages 32-45 for enpp7. All embryos were analysed from the same experimental set. The differences in colours result from clearing the embryos and photographing them against a white background to visualise staining. (A) Whole-mount in situ hybridisation analysis of enpp1 expression. Lateral view at stages 28 (i), 35/36 (ii) and 37/38 (iii). Detail of the fin at stage 41 (iv) and dorsal view of a stage 41 embryo (v). Anterior is right and dorsal is up, except in (v). da, dorsal aorta; dc, duct of cuvier; dlav, dorsal longitudinal anastomosing vessel; h, heart; pcv, posterior cardinal vein; ps, pronephric sinus; vbi, ventral blood island. (B) Whole-mount in situ hybridisation analysis of enpp4 expression. Animal view at stage 5 (i), anterior view at stage 22 (ii), lateral view at stages 32 ...
In article ,4dgqf7$10g0 at majestix.uni-muenster.de, Torsten Boerchers ,borcher at uni-muenster.de, writes: ,Torsten Boerchers ,borcher at uni-muenster.de, wrote: ,,Forwarded from rhubner at molbiol.ox.ac.uk: ,, ,,hello Torsten, ,, although you dont give details about those multiple bands I would ,,suspect either premature termination or/and read-through (not completely ,,linearized... ,,who can COMPLETELY linearize plasmids?) of the T7 pol beyond the RE ,site , ,,,multiple bands depending on run-on transcription intensity... ,,wouldnt cutting with a second RE help troubleshooting? but, what ,,then ? (i.e., how to stop such rolling-circles efficiently?). In the ,case of premature termination I fear nothing special can be done... ,,oh yes, you might also have alternative initiation (change into other ,RNA pol vector to check? can be predicted by seq analysis to some ,extend...) ,, Obviously _I_ too need to read/hear/experience more about T7 pol ,,transcription initiation/termination... any ...
so what are you making the probes for, and what kind of probes are they? because if your insert isnt HUGE, you can always PCR the insert out (using primers that anneal outside of the T7 and Sp6 sites so that they are included in your PCR product), then use the PCR product for your transcription reaction. I do this to make T7 and Sp6 RNA probes for in situ hybridisations and it works really well.... ...
Digoxigenin (DIG)-labeled RNA antisense probes are widely used for in situ hybridization due to their high sensitivity and specificity. DIG-labeled RNA probes are also stable for more than a year, making them ideal for long-term studies with high consistency and low technical variation. In this application note read how DIG-labeled probes were used to investigate the localization of human hepatocyte growth factor (hHGF), a cell surface cytokine binding the c-MET-receptor, implicated in the tyros
Non-radioactive biotinylated RNA probes specific for plus (+) and minus (-) sense RNAs of brome mosaic virus (BMV) were synthesized in vitro from a plasmid bearing a 200 base pair fragment complementary to the 3 terminus of each of the three genomic RNAs of the virus. Using virion RNAs isolated from BMV infected barley plants, the sensitivity of biotinylated RNAs as hybridization probes was compared with that of 32P-labeled probes in Northern hybridization assays. Although the sensitivity of biotinylated and 32P-labeled probes is similar (approximately 5 pg), the time required to detect the RNA bands was much less than for autoradiography; (-) sense RNAs could be detected in 30 min whereas 48 h or more were required by autoradiography. The value of biotinylated probes for following RNA stability was exemplified by the detection of supplied inocula in protoplasts 24 h post-inoculation. Quantitation of relative accumulation of progeny (+) and (-) sense RNAs by densitometry of the Northern blots ...
The RNA probe was in vitro transcribed with the recombinant of the Y-chromosome specific deoxyribonucleic acid (DNA) sequence and transcription-vector Bluescript SK M13+. The preparation of the RNA probe is not difficult, but it does require a subcloning procedure which is unnecessary in the preparation of the DNA probe. The results indicate the superiority of the RNA probe over the previously studied DNA probe. The RNA probe has a high hybridization efficiency which increases the sensitivity of the test and shortens the period of time necessary for obtaining the resulting stains. 2 figures, 7 references. (Author abstract modified) ...
This protocol is designed for In Situ Hybridization and colorimetric detection of digoxigenin-labeled RNA probes on paraformaldehyde fixed cryosectioned tissue. This protocol has primarily been tested on mouse embryos and on brain tissue and takes approximately three days. ...
The ribonuclease protection assay (RPA) is a highly sensitiveand specific method for the detection and quantitationof mRNA species. The assay was made possibleby the discovery and characterization of bacteriophageencodedDNA-dependent RNA polymerases (SP6 T7and T3).,RNase,Protection,Assay,(RPA),,,Using,DIG-Labeled,RNA,Probes,biological,advanced biology technology,biology laboratory technology,biology device technology,latest biology technology
It is estimated that metastasis is the main reason in about 90% of cancer deaths. Therefore, a better understanding of the key molecular players in determining and regulating this process is urgently needed. Previous studies in fixed cells in culture have shown, that certain microRNA (miRNA) molecules are in significantly higher numbers in exosomes of highly metastatic cells in comparison to low metastatic cells [1]. However, in real patient tissues, issues such as the cell heterogeneity in the primary tumor and the definition of metastatic clones are a major challenge in the context of metastasis.. In this project, we used human colon tissue sections, which are relevant samples for routine diagnosis in pathology, to study the chromatin nanostructure of different cells types and the presence and distribution of five metastatically relevant miRNAs (miR-21, miR-31, mi-R210, miR-135b and miR-218).. DNA-sequences complementary to the above-mentioned metastatically relevant miRNA were synthesized and ...
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The ability to visualize the expression of a gene in both time and space is an essential tool of developmental biology. Here, we detail a robust method for in situhybridization of RNA probes to...
Traces of DEPC modify purine residues (A+G) in RNA by carboxymethylation. Therefore, DEPC must always be removed from solutions or containers by autoclaving or heating at 100°C for 15 min. Cell-free translation of carboxymethylated RNA will yield less protein than with unmodified template. Hybridisation is not seriously affected unless the RNA probe is heavily ...
5-(3-aminoallyl)uridine 5-triphosphate (AA-UTP) is a cost-effective alternative to direct incorporation of a labeled-UTP when preparing non-radioactive RNA probes by in vitro transcription. AA-UTP is a good substrate for T7, T3 and SP6 RNA polymerases to
IL-2-regulated genes in PBMCs. Pre-activated, rested human PBMCs were left untreated or restimulated for 4 hr with IL-2, and RNA probes were prepared for hybrid
Kit Component:- KN207072G1, BRUNOL5 gRNA vector 1 in pCas-Guide vector- KN207072G2, BRUNOL5 gRNA vector 2 in pCas-Guide vector- KN207072D, donor…
A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during in situ hybridization. RNA probes can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter. Some bacterial viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts inserted in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene. Since James Watson and Francis Crick revealed the double helix nature of DNA molecule(Watson & Crick, 1953), the hydrogen bonds between the four bases are well known: adenine always binds to thymine and cytosine always binds to guanine. This binding pattern is the basic principle of modern genetic technologies. Joseph Gall and Mary Lou Pardue published a paper in 1969 demonstrating that radioactive marked ribosomal DNA can be used to detect its ...
Purpose: : Musashi (MSI), an mRNA binding protein, has been shown to be expressed in neural progenitor cells. (reviewed by Okano et al., 2005) The goal of this study was to determine the expression of MSI in the chick retina, with special emphasis on putative stem cells within the retina. Methods: : Immunohistochemistry and in situ hybridization were performed with 14 um sections of E3, E4, E5, E6, E8 and E18 eye using anti-MSI antibody and a MSI specific Digoxigenin-labeled RNA probe. Results: : MSI appeared to be expressed in the presumptive amacrine and photoreceptor cells of the developing retina, the ciliary margin zone where stem cell preside, and in the lens epithelium. Conclusions: : MSI labels more than stem cells in the chick retina. ...
Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments. The PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ target accessibility patterns. The different types of sequence
ePIC (electronic Publication Information Center) is the official repository for publications and presentations of Alfred Wegener Institute for Polar and Marine Research (AWI)
Tissue preparation. Twenty adult male Sprague Dawley 20 rats (100-120 gm body weight) were anesthetized with choral hydrate (3.5 mg/100 gm body weight) and perfused transcardially with a solution of 4% paraformaldehyde in 0.1 m phosphate buffer (PB), pH 7.3. Brains were post-fixed overnight, rinsed with PB, and sequentially transferred to 12%, 14%, and 16% sucrose solutions. Brains were then frozen on dry ice, and sections of 30-40 μm thickness were obtained on a cryostat.. Probe preparation. [35S]- and [33P]-labeled sense and antisense RNA probes were generated from a 730 basepair (bp) PstI insert (corresponding to nucleotides 1500-2230 of the rat 5HT3R cDNA), using the TransProbe T Kit (Pharmacia, Piscataway, NJ). The [35S]- and [33P]-labeled antisense probes were synthesized separately.. In situ hybridization-immunocytochemistry labeling. In situ hybridization combined with immunolabeling was performed as described previously (Morales et al., 1996a). Free-floating cryosections were incubated ...
Hi all. Is there anybody who could advice me a reliable protocol for whole mount in situ hybridization with organs dissected from the last instar larva, e.g. salivary , gut, Malpighian, etc. ? I am especially interested in the fixation step and necessity and extend of proteinase treatment. I am using DIG RNA probe. Please respond directly by e-mail to mach at entu.cas.cz. Thanks for your help. Vaclav Mach Institute of Entomology Czech Republic ...
THOMAS:. Wash. Rock. Aspirate. Repeat.. Six weeks of diligent work had culminated with six delicately miniscule mouse embryos, so transparent that my eyes often lost track of them as they sloshed around in solution. My mentor and I had spent the past month running PCRs, conjuring up gels, and transforming plasmids into bacteria, all in preparation for this final step: the in situ hybridization. The purpose of this complicated sounding process was to insert RNA probes into the embryos, which would allow us to observe the expression pattern of the gene Fgf12 in embryonic heart development (specifically in the atrium). The next four days would be filled with myriads of strange solutions comprised of mysterious, unpronounceable substances. These deceptively clear liquids were capable of performing painstaking chemical processes at the molecular level, something that my mind could hardly fathom. Each step involved washing the embryos with different solutions (which could contain anything from ...
Fig. 5 pax-6 is expressed in limited regions of the neural keel. Paraffin sections of embryos were hybridized with 35S-labelled RNA probes (probe A, probe C gave the same results). (A,B) Parasagittal sections of a 14 h embryo. Expression is seen in the optic vesicle (A, B), the prospective forebrain (B) and the prospective hindbrain and spinal cord (A, B). Lines in B indicate the plane of section in panels C and D. (C) Cross-section of the prospective forebrain at 14 h. Expression is seen in the dorsal half, excluding the dorsal-most cells of the forebrain. (D) Cross-section of the neural keel at 14 h. Expression is seen in the ventral half. (E) Parasagittal section of the optic vesicle (ov) at 14 h. Expression is seen in the ventral and lateral part of the vesicle. Little or no expression is detectable in the dorsal part. Expression is also seen in the ectoderm (ect) overlying the optic vesicle. (F) Horizontal section of an 18 h embryo. Expression is seen in the diencephalon (di) and the ...
Adding T7 promoter sequence to vector - posted in Molecular Biology: Hi all:I am trying to make RNA probe for ISH. I ordered cDNA clone from a company and I realized that the vector they used does not have T7 or T3 or SP3 promoter sequence. So, how can I add the promoter sequence to my vector? Also, I streaked the glycerol stock which I got yesterday but no colonies today, what could have gone wrong?Vector:pME18S-FL3Thanks in advance
Buy our Recombinant Human BRUNOL6 protein. Ab163736 is a full length protein produced in Wheat germ and has been validated in WB, ELISA. Abcam provides free…
According to the authors, Chick cDNAs of Slit1 (a 1061-bp RT-PCR fragment from a chick cDNA library corresponding to nt 605 1665 of mSlit1, GenBank accession no. AF144627)...was used to generate digoxigenin or S35-labeled riboprobes... The sequence below was obtained from NCBI (AF144627) using the information provided ...
We have investigated the usefulness of serum hepatitis delta virus (HDV) RNA detection using a slot hybridization analysis of serum samples from ten patients with acute hepatitis and delta markers (group I), from 28 patients with chronic delta hepatitis (group II) and from seven liver graft recipients with hepatitis B virus (HBV) and HDV related cirrhosis or fulminant hepatitis (group III). The slot-blots were hybridized with both HDV-complementary DNA and single-stranded RNA probes. With the single-stranded RNA probe, HDV RNA was detected in the first serum sample available in 9/10 of the patients with acute hepatitis (group I). In addition, HDV RNA was detected in 8/9 and 7/8 of the samples obtained within and after 1 month of the onset of hepatitis. Five of the ten patients scored positive for HDV RNA and negative for hepatitis delta antigen (HDAg) while one was negative for HDV RNA and positive for HDAg. The same RNA probe enabled the detection of serum HDV RNA in 21/28 chronic hepatitis ...
RNA analysis products include RNA purification kits, RNase protection (RNasin RNase Inhibitor), RNA quantification dyes and fluorometers, and RT-PCR kits. We also offer systems for RNA synthesis (in vitro transcription), including production of RNA probes (Riboprobes).
Array Hybridization and Detection of Fluorescence was performed as follows. 12 ng of IVT was fragmented in 40 mM Tris-actetate, pH 8.0, 100 mM potassium acetate, and 30 mM magnesium acetate for 35 minutes at 94 °C. The fragmented, labeled RNA probes were diluted in hybridization buffer at a final composition of 1 x 2-NMorpholinoethanesulfonic acid (MES (buffer (pH 6.5), 50 pM Bio948 (control biotinylated oligo that hybridizes to landmark features on the probe array (Genetics Institute, Cambridge, MA)), 100 u.g/ml herring sperm DNA (Promega, Madison,WD, 500 u.g/ml acetylated BSA (Invitrogen Life Technologies) and 1 jul/jag standard curve reagent (Proprietary reagent supplied by Gene Logic, Gaithersburg, MD). This hybridization solution was pre-hybridized with two glass beads (Fisher Scientific, Pittsburgh, PA) at 45 °C overnight. The hybridization solution was removed to a clean tube, heated for 1-2 min at 95 °C and microcentrifuged on high for 2 minutes to pellet insoluble debris. Affymetrix ...
A method was developed for enriching the concentration of mycoplasmalike organism (MLO) DNA in diseased plant extracts. With this method, fragments of DNA of the aster yellows (AY) MLO were cloned in pSP6 plasmid vectors and amplified in Escherichia coli strain JM83. Labeled double-stranded DNA probes were prepared by nick translation of recombinant plasmids by using either 32P nucleotides or biotinylated nucleotides. Single-stranded RNA probes (riboprobes) were synthesized in vitro by using SP6 polymerase and linearized recombinant plasmids and were labeled with 32P. The probes all hybridized with nucleic acid from AY MLO-infected, but not healthy, plants. Some hybridized also with nucleic acid from plants infected by other MLOs. Hybridization patterns indicated existence of a cluster of MLOs, including AY MLO, that share greater nucleotide sequence homology with one another than with other MLOs. Riboprobes were used to differentiate AY MLO from others in the AY MLO cluster.. Additional ...
Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19. ...
Definition : Molecular assay reagents consisting of a known fragment of nucleic acid, typically labeled with a tag. Probes may be classified according to their chemical structure as DNA or RNA probes, strandedness as double- or single-stranded (ds and ss, respectively), and origin as cloned (recombinant), genomic, or synthesized (oligonucleotides). Probes may be radioactively or nonradioactively (both enzymatic and nonenzymatic) labeled. Nucleic acid probes are used in tests to obtain information about the genetic characteristics of a sample; these procedures are generically known as nucleic acid assays and include hybridization assays, amplification methods (e.g., polymerase chain reaction), and electrophoretic separation.. Entry Terms : Probes, Nucleic Acid , Nucleic Acid Probes , Reagents, Molecular Assay, Nucleic Acid Probe. UMDC code : 19666 ...
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Hunted down! Dramatic moment a notorious gang of ivory poachers are caught in an undercover sting in Zambia: Photo documentation of the dramatic moment notorious ivory poachers are caught - read more.. How Lizard Genitalia Became a Black Market Craze: Monitor lizards were poached for their meat and skins… now theyre dying for their penises - read more.. Whale Strikes and Kills Canadian Rescuer After He Helps Free It: Marine community mourns the loss of Joe Howlett, dedicated whale rescuer who died after successfully disentangling a whale - read more.. Sunday Night With Megyn Kelly Team Travels to Kenya to Report on Efforts to Protect Elephants - read more.. ...
The ability of labeled DNA or RNA probes to bind with high affinity and specificity to complementary nucleic acid sequences forms the basis of nucleic acid hybridization assays. Immunoassays, on the other hand, exploit the strong and specific interaction between antigens and antibodies. One aspect of this dissertation deals with enhancement of the sensitivity of bioluminescence hybridization assays based on the photoprotein aequorin. This is achieved by introducing, enzymatically, multiple aequorin labels per DNA hybrid. The bound aequorin is determined by its characteristic bioluminescence. An 8-fold improvement in sensitivity is observed with the amplified assay as compared to an assay without the amplification step. Another aspect of this dissertation involves the development of two novel microtiter well-based DNA hybridization assays in which an expressible cDNA fragment encoding firefly luciferase serves as a reporter molecule. The reporter molecule undergoes in vitro expression through coupled
We plan to apply the methodologies developed from our current NMFS-funded project for mtDNA markers, DNA fingerprinting, RNA probes and PCR technologies to quantify low-levels of Perkinsus marinus infections in order to fully describe the etiology of this oyster disease. Our progress in working with genetic markers of P. marinus has: Developed a sensitive PCR-based diagnostic assay for detecting P. marinus infections ; Identified an extrachromosomal relic plastid-genome in P.
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つい引きずっている悪い習慣を新たな視点から捉え直すことで、その悪習を断ち切れるとしたらどうでしょう。 精神科医ジャドソン・ブルワーは、喫煙から過食に至るまで、よくないと自覚していても、どうしてもやめられない習慣、つまり依存症と「マインドフルネス」との関係を研究しています。習慣形成のメカニズムをまず詳しく学び、その知識をもとに、簡単に実行できるのに実は深い意味のある、悪習への対策を見つけましょう。今度こそ、つい一服、ほんの一口、車の運転中に一瞬だけメール…という誘惑に勝てるかもしれません。
TY - JOUR. T1 - Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. AU - Melton, D. A.. AU - Krieg, P. A.. AU - Rebagliati, M. R.. AU - Maniatis, T.. AU - Zinn, K.. AU - Green, M. R.. N1 - Funding Information: ACKNOWLEDGEMENTS We are grateful to D. Ward and B. Mierendorf for sharing their unpublished results. We thank K. Breakey for help in preparing figures. P. K. acknowledges support from the Fogarty International Fellowship program. M.R. is grateful for support from an NSF Predoctoral Fellowship. This work was supported by a grant from the NIH to T.M. and grants from the NIH and the Chicago Community Trust/Searle Scholars Program to D.M. Copyright: Copyright 2010 Elsevier B.V., All rights reserved.. PY - 1984/9/25. Y1 - 1984/9/25. N2 - A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the ...
Figure 4. Localization of GRK7 mRNA in ground squirrel retinas using in situ hybridization.. 35S-labeled antisense and sense RNA probes were produced from the 146-bp fragment corresponding to nucleotide sequence 1019-1164 by in vitro transcription and hybridized to cryostat sections of ground squirrel retina as described in the Methods. (A) Dark field image of ground squirrel retina hybridized with an antisense probe for GRK7. Retinal pigment epithelium (RPE), photoreceptor cell layer (PCL), inner segment layer (INL), ganglion cell layer (GCL). (B) Dark field image of ground squirrel retina hybridized with a sense probe for GRK7. Abbreviations are as described in (A). Since the retinal pigment epithelium is black, it appears white in dark field images.. ...
Samples Cervical swab specimens, which were obtained for screening test of cervical cancers, were collected from a total 146 women at Seoul St. Marys Hospital between January and December of 2011. All specimens were collected, placed in a ThinPrep PreservCyt solution (Hologic Inc., Malborough, MA, USA) and a SurePath solution (BD, Franklin Lakes, NJ), and were then stored at -70°C until assayed for HPV tests. All specimens were classified into 4 groups according to their cytologic results: negative (n=62), ASCUS (n=39), LSIL (n=35) and HSIL (n=10).. HC2 test HPV DNA testing by the HC2 test was performed with an HC2 assay system according to the manufacturers protocol (Qiagen Inc.,Valencia, CA, USA). The specimens were denatured at 65°C for 45 minutes and hybridized under high-stringency conditions with a mixture of RNA probes that detect 13 different oncogenic HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. The resultant DNA-RNA hybrids were captured on the surface of the ...
The members of the IgLON superfamily of cell adhesion molecules facilitate fundamental cellular communication during brain development, maintain functional brain circuitry, and are associated with several neuropsychiatric disorders. Usage of alternative promoter-specific 1a and 1b mRNA isoforms in Lsamp, Opcml, Ntm and the single promoter of Negr1 in the mouse and human brain has been previously described. To determine the precise spatiotemporal expression dynamics of Lsamp, Opcml, Ntm isoforms and Negr1, in the developing brain, we generated isoform-specific RNA probes and carried out in situ hybridization in the developing (embryonic, E10.5, 13.5, 17; post natal, P0) and adult mouse brains. We show that promoter-specific expression of IgLONs is established early during pallial development (at E10.5), where it remains throughout its differentiation through adulthood. In the diencephalon, midbrain and hindbrain, strong expression patterns are initiated a few days later and begin fading after birth,
TY - JOUR. T1 - 2-O-alkyl oligoribonucleotides as antisense probes. AU - Iribarren, A. M.. AU - Sproat, B. S.. AU - Neuner, P.. AU - Sulston, I.. AU - Ryder, U.. AU - Lamond, A. I.. PY - 1990/10/1. Y1 - 1990/10/1. N2 - 2-O-Methyl oligoribonucleotides have recently been introduced as antisense probes for studying RNA processing and for affinity purification of RNA-protein complexes. To identify RNA analogues with improved properties for antisense analysis, 2-O-alkyl oligoribonucleotides were synthesized in which the alkyl moiety was either the three-carbon linear allyl group or the five-carbon branched 3,3-dimethylallyl group. Both these analogues were found to be completely resistant to degradation by either DNA- or RNA-specific nucleases. Use of biotinylated derivatives of the probes to affinity-select ribonucleoprotein particles from crude HeLa cell nuclear extracts showed that the presence of the bulky 3,3-dimethylallyl group significantly reduces affinity selection, whereas the allyl ...
[email protected] wrote: Hello Histonetters, I would like to do fluorescence in situ hybridization on paraffin embedded sections using RNA probes. I would appreciate it very much if anybody who has worked on this could share the protocol with me. Thanks in advance. Agripina Agripina C. Suarez Cardiovascular Research Laboratory McDonald Research Wing, St. Pauls Hospital 1081 Burrard St., Vancouver B.C., Canada V6Z 1Y6 Phone: (604) 631-5659 Fax: (604) 631-5351 Hi Agripina, I would suggest the Hyb-Probe system from Shandon Lipshaw Cat.#502100 this system utilizesFluorescein-labeled nucleic acid probes. This system allow identification of specific nucleic acids DNA and RNA. This system is very time efficient and sensitive. Good luck and I Hope this info. helps. Sincerely, Carol Tucker-Burden Emory University Atlanta, GA. Dept. Of Surgery Transplant Immunology Research Ctr. ____________________________________________________________________ More than just email--Get your FREE Netscape ...
In this report, we show that the products of UL47, UL49, and US11 ORFs bind RNA in vitro and in the context of infected cells, and that the packaged RNAs can be expressed in infected cells. We also show that VP22, the product of the UL49 ORF, mediates the transfer of the RNA from cell to cell. Relevant to our results are the following:. (i) The procedure we have used to identify the protein capable of binding RNAs was to electrophoretically separate virion proteins in denaturing gels, renature the proteins in situ, and react them with a labeled riboprobe representing the RNA detected in all virion preparations tested. Using this procedure, we unambiguously demonstrated that three virion protein bands bind RNAs. These proteins were identified as the products of the UL47, UL49, and US11 genes. In these assays, we used as probe the most abundant RNA packaged in virions. Because we used a riboprobe representing a single viral RNA, we cannot exclude the possibility that there exist virion proteins ...
Expression of Raldh1. In situ hybridisation was performed on wholemount embryos with DIG- or fluorescein-labelled riboprobes. Unless otherwise stated, rostral i
Authors said: For in situ hybridization, antisense probes for cathepsin D, B, and L (Zuzarte-Luis et al.,[2007]), where they said:cCatD fwd: 5′-TTC TGC GCT TCT GCT TTA GGG-3′ and rev: 5′-TGA GTG GGT TTC TAA TCC TGA-3. The sequences presented was excerpted from the full length using these sequences ...
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SOLIScript 1-step Probe Kit is suitable for ROX-dependent and ROX-independent qPCR cyclers. Learn about the product and order free samples here!