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At least 109 records · Page 6Linked to original sources
PubMed2006

RNA extraction for arrays.

DNA microarrays enable insights into global gene expression by capturing a snapshot of cellular expression levels at the time of sample collection. Careful RNA handling and extraction are required to preserve this information properly, ensure sample-to-sample reproducibility, and limit unwanted technical variation in experimental data. This chapter discusses important considerations for "array-friendly" sample handling and processing from biosamples such as blood, formalin-fixed, paraffin-embedded samples, and fresh or flash-frozen tissues and cells. It also provides guidelines on RNA quality assessments, which can be used to validate sample preparation and maximize recovery of relevant biological information.

Animals↗
PubMed2003

RNA extraction and RT-PCR procedures adapted for the detection of enterovirus sequences from frozen and paraffin-embedded formalin-fixed spinal cord samples.

Methods for detecting enterovirus RNA in both paraffin-embedded, formalin-fixed and frozen spinal cord sections from amyotrophic lateral sclerosis (ALS) patients were established. A proteinase K digestion following the deparaffinization procedure was required for the fixed spinal cord sections, whereas only one step of crushing in phosphate buffered saline was necessary for the frozen samples prior to the extraction of the RNA. With an optimized reverse transcription and PCR procedure, enterovirus RNA could be detected from frozen and fixed archival spinal cord samples.

Amyotrophic Lateral Sclerosis↗
PubMed2002

Cytoplasmic RNA extraction from fresh and frozen mammalian tissues.

The quality of collections of expressed sequence tags andfull-length cDNAs is adversely affected by the presence of "junk" clones derivedfrom unspliced or partially spliced RNAs present in conventional total RNA preparations. One can overcome this problem by using intact cytoplasmic RNA to create cDNA libraries, but the methods in the literature that describe the preparation of RNA only work well for extracting cultured cells. Cell lines are not as diverse as one would like, and to clone comprehensive sets of human and model organism full-length cDNAs, libraries have to be prepared from tissue samples. Thus, we have developed a robust and inexpensive method that allows intact cytoplasmic RNA to be extracted from both fresh and frozen mammalian tissues. A mouse full-length, cap-trapped cDNA library prepared with RNA using this new procedure had excellent characteristics.

Animals↗
PubMed1997

Analysis of T cell receptor beta chain CDR3 size using RNA extracted from formalin fixed paraffin wax embedded tissue.

AIMS: To isolate RNA and DNA simultaneously from formalin fixed paraffin wax embedded tissue to assess the clonality of enteropathy associated T cell lymphomas and to analyse it in detail by a non-radioactive method of T cell receptor complementarity determining region 3 (CDR3) spectratyping. METHODS: DNA and RNA were isolated simultaneously from formalin fixed paraffin wax embedded tissue blocks and subjected to the polymerase chain reaction (PCR) and semi-nested reverse transcription PCR (RT-PCR), respectively. The RT-PCR T cell receptor V beta products were analysed by CDR3 spectratyping using a denaturing polyacrylamide gel and silver staining. RESULTS: Usable DNA and RNA were isolated simultaneously from formalin fixed paraffin wax embedded tissue. The specific clonality of the tissue was successfully analysed by a non-radioactive method of T cell receptor CDR3 spectratyping of the RT-PCR products. CDR3 spectratying of the RT-PCR products demonstrated the precise clonal nature of the tumour and non-tumour tissue showing that the non-tumour tissue comprised an oligoclonal population of a number of different T cell receptor V beta families. The tumour tissue comprised two T cell subtypes of the one family, T cell receptor V beta 9. CONCLUSIONS: RNA and DNA were isolated from formalin fixed paraffin wax embedded enteropathy associated T cell lymphoma tissue. Detailed analysis of clonality can be carried out by a non-radioactive method of CDR3 spectratyping.

DNA, Neoplasm↗
PubMed1995

[Comparative studies and application of a simple method of extraction of RNA from hepatitis C virus].

Conventional RNA extraction methods, such as SDS/phenol and AGPC methods involve time consuming and complicated manipulation procedures, which could be a cause of contamination. In this study, we compared the conventional methods with a method using a newly developed reagent, "SepaGene-RV" to extract RNA. The results obtained by the new method were the same as those obtained by the conventional methods in the comparative experiments with the sera of patients infected with HCV and detection limit tests using recombinant HCV genome. The sensitivity to detect HCV-RNA with SepaGene-RV was equivalent to that of conventionally used methods to extract RNA, and the manipulation procedure was simpler and it was less time consuming. In addition, we could detect plus-stranded and minus-stranded HCV-RNA by polymerase chain reaction assay (PCR) from liver tissue obtained from liver biopsy and peripheral blood mononuclear cells (PBMC). We confirmed that HCV infects not only liver tissue but also PBMC where replication occurs.

Genome, Viral↗
PubMed1965

Induction in vitro of antibodies to phage T2: antigens in the RNA extract employed.

Cells from rat peritoneal exudate incubated in vitro with phage T2 yield RNA which induces cultures of rat lymph nodes to form antibodies. Complement fixation reveals T2 head, tail, and internal protein antigens in the RNA. Inoculating the RNA into mice that had previously been stimulated primarily with whole phage T2 results in a secondary response of neutralizing antibody.

Animals↗
PubMed1988

Characterisation of messenger RNA extracted post-mortem from the brains of schizophrenic, depressed and control subjects.

Messenger RNA, obtained from post-mortem brain of 10 schizophrenics, five depressed patients and 10 control subjects, was characterised with respect to a number of parameters. It was found that post-mortem delay was not the major factor in determining RNA yield, size (as determined by cDNA synthesis) and biological activity. Biological activity, as determined by in vitro translation in a reticulocyte-lysate system, could be observed using messenger RNA from periods of 0 to 84 hours post-mortem. Two-dimensional gel analysis of the newly-synthesised radiolabelled products obtained from this material revealed several hundred individual species but no consistent degradation of any particular species with post-mortem delay. It is suggested, therefore, that premortem changes are as important as post-mortem changes in determining RNA yield, size and biological activity. Although no consistent difference could be found between patients and controls using any of these parameters, this study confirms that, by isolating messenger RNA from post-mortem human brain, valuable information can be gained on gene expression in psychiatric disorders.

Brain↗