scholarly journals Benefits of random-priming: Exhaustive survey of a cDNA library from lung tissue of a SARS patient

2011 ◽  
Vol 83 (4) ◽  
pp. 574-586
Author(s):  
Hongkai Wu ◽  
Jinwen Wang ◽  
Riqiang Deng ◽  
Ke Xing ◽  
Yuanyan Xiong ◽  
...  
Keyword(s):  
Cdna Library ◽  
Lung Tissue ◽  
Random Priming

scholarly journals Poly(A) Polymerase Modification and Reverse Transcriptase PCR Amplification of Environmental RNA

2005 ◽  
Vol 71 (3) ◽  
pp. 1267-1275 ◽  
Author(s):  
Lina M. Botero ◽  
Seth D'Imperio ◽  
Mark Burr ◽  
Timothy R. McDermott ◽  
Mark Young ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Cdna Library ◽  
Cdna Clone ◽  
Pcr Amplification ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
Random Priming ◽  
Rna Targeting ◽  
Pure Cultures ◽  
Heated Soil

ABSTRACT We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing Bacteria and Eukarya taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and Bacteria-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the Firmicutes and the α-Proteobacteria. Unexpectedly, there were no Archaea clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and Archaea-biased primers and unmodified RNA demonstrated the presence of novel Archaea in the soil. Experiments with pure cultures of Sulfolobus solfataricus and Halobacterium halobium revealed that some Archaea rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems.

Isolation of Asbestos from Lung Tissue and Sputum

1982 ◽  
Vol 40 ◽  
pp. 330-331
Author(s):  
M. G. Williams ◽  
C. Corn ◽  
R. F. Dodson ◽  
G. A. Hurst
Keyword(s):  
Sodium Hypochlorite ◽  
Lung Tissue ◽  
Body Fluids ◽  
Unknown Etiology ◽  
The Past

During this century, interest in the particulate content of the organs and body fluids of those individuals affected by pneumoconiosis, cancer, or other diseases of unknown etiology developed and concern was further prompted with the increasing realization that various foreign particles were associated with or caused disease. Concurrently particularly in the past two decades, a number of methods were devised for isolating particulates from tissue. These methods were recently reviewed by Vallyathan et al. who concluded sodium hypochlorite digestion was both simple and superior to other digestion procedures.

In situ microprobe quantitation of inorganic particle burden in paraffin sections of lung tissue

1988 ◽  
Vol 46 ◽  
pp. 990-991
Author(s):  
Jerrold L. Abraham
Keyword(s):  
Lung Tissue ◽  
Quantitative Information ◽  
Particulate Material ◽  
Paraffin Sections ◽  
Tissue Samples ◽  
Qualitative And Quantitative ◽  
The Past ◽  
Quantitative Analyses ◽  
Data Result

Inorganic particulate material of diverse types is present in the ambient and occupational environment, and exposure to such materials is a well recognized cause of some lung disease. To investigate the interaction of inhaled inorganic particulates with the lung it is necessary to obtain quantitative information on the particulate burden of lung tissue in a wide variety of situations. The vast majority of diagnostic and experimental tissue samples (biopsies and autopsies) are fixed with formaldehyde solutions, dehydrated with organic solvents and embedded in paraffin wax. Over the past 16 years, I have attempted to obtain maximal analytical use of such tissue with minimal preparative steps. Unique diagnostic and research data result from both qualitative and quantitative analyses of sections. Most of the data has been related to inhaled inorganic particulates in lungs, but the basic methods are applicable to any tissues. The preparations are primarily designed for SEM use, but they are stable for storage and transport to other laboratories and several other instruments (e.g., for SIMS techniques).

The Identification of Phospholipid Micelles in Glutaraldehyde-Urea Embedded Tissue

1975 ◽  
Vol 33 ◽  
pp. 494-495
Author(s):  
Daniel C. Pease
Keyword(s):  
Electron Micrographs ◽  
Lung Tissue ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Type Ii ◽  
Lead Citrate ◽  
Phospholipid Micelles ◽  
Type Ii Pneumocytes ◽  
Ultrathin Sections ◽  
Polar Water

It is reasonable to think that phospholipid micelles should be visible and identifiable in electron micrographs of ultrathin sections if only they can be preserved throughout the embedding process. The development of highly polar, water-containing, aminoplastic embedments has made this a likely possibility. With this in mind, an investigation of the lecithin-secreting, Type II pneumocytes of the lung is underway.Initially it has been easiest to recognize phospholipid micelles in lung tissue fixed first with glutaraldehyde, and then secondarily exposed to osmium tetroxide. However, the latter is not a necessary concomitant for micellar preservation. Conventional uranyl acetate and lead citrate staining is finally applied. Importantly, though, the micelles have been most easily seen in tissue embedded in 507. glutaraldehyde polymerized with urea, as described in detail by D.C. Pease and R.G. Peterson (J. Ultra- struct. Res., 41, 133, 1972). When oriented appropriately, the micellar units are seen as tiny, bilayer plates.

Biomedical applications: Pitfalls in the practice

1994 ◽  
Vol 52 ◽  
pp. 378-379
Author(s):  
J. D. Shelburne ◽  
Peter Ingram ◽  
Victor L. Roggli ◽  
Ann LeFurgey
Keyword(s):  
Operating Room ◽  
Liquid Nitrogen ◽  
Lung Tissue ◽  
Biomedical Applications ◽  
Microprobe Analysis ◽  
Tissue Sample ◽  
Cellular Level ◽  
Tissue Samples ◽  
Asbestos Fibers

At present most medical microprobe analysis is conducted on insoluble particulates such as asbestos fibers in lung tissue. Cryotechniques are not necessary for this type of specimen. Insoluble particulates can be processed conventionally. Nevertheless, it is important to emphasize that conventional processing is unacceptable for specimens in which electrolyte distributions in tissues are sought. It is necessary to flash-freeze in order to preserve the integrity of electrolyte distributions at the subcellular and cellular level. Ideally, biopsies should be flash-frozen in the operating room rather than being frozen several minutes later in a histology laboratory. Electrolytes will move during such a long delay. While flammable cryogens such as propane obviously cannot be used in an operating room, liquid nitrogen-cooled slam-freezing devices or guns may be permitted, and are the best way to achieve an artifact-free, accurate tissue sample which truly reflects the in vivo state. Unfortunately, the importance of cryofixation is often not understood. Investigators bring tissue samples fixed in glutaraldehyde to a microprobe laboratory with a request for microprobe analysis for electrolytes.

Analysis of cDNA library of Cucumis sativus L. challenged by Pseudomonas syringae pv. Lachrymans

2009 ◽  
Vol 31 (10) ◽  
pp. 1042-1048
Author(s):  
Guan-Jun LIU ◽  
Li-Juan WANG ◽  
Zhi-Wei QIN ◽  
Ling-Bo MENG
Keyword(s):  
Cdna Library ◽  
Cucumis Sativus ◽  
Pseudomonas Syringae
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