Linked Life Data

12019773

Source:http://linkedlifedata.com/resource/pubmed/id/12019773

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
2002-5-21
pubmed:abstractText
Quantitation of the level of specific mRNA involves the isolation of total RNA or poly(A)+ RNA as a starting materiaL Thus, this result is the sum of the transcription and degradation of mRNA. Here we report a rapid, sensitive, and high-throughput methodology for gene expression analysis from nuclear poly(A)+ RNA via the reduction of the cytosolic components. The cells were first trapped on the glass fiber membranes of 96-well filter plates and subsequently exposed to non-ionic detergent to achieve cell membrane permeation. The cytosolic components, which contain preexisting mRNA, were removed by washing with the appropriate buffer, while nuclei remained in the filter plates. Lysis buffer was then used to release nuclear mRNA, which was collected on oligo(dT)-immobilized PCR plates for the capture of poly(A)+ RNA, on which RT-PCR was performed. The reduction of the cytosolic components and the preservation of the nuclear components were confirmed by electron microscopy, agarose gel electrophoresis, PCR of mtDNA, and RT-PCR of pre-splicing immature beta-actin poly(A)+ RNA. Using this method, we clearly identified UVC-induced p21 gene expression that is not detectable with conventional whole cell methods.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0736-6205
pubmed:author
pubmed:issnType
Print
pubmed:volume
32
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1014-6, 1018, 1020
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Gene expression analysis from nuclear Poly(A) RNA.
pubmed:affiliation
University of California, Irvine, USA.
pubmed:publicationType
Research Support, Non-U.S. Gov't, Technical Report