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Results for “RNA quality”

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At least 37 records · Page 2Linked to original sources
PubMed2003

A two-step method for the extraction of high-quality RNA from endoscopic biopsies.

The use of molecular techniques such as quantitative RT-PCR depends on the quality of cellular RNA. In particular, RNA extraction from endoscopic biopsies is difficult with respect to yield, and especially integrity. Endoscopic biopsies taken from the gastric antrum, corpus and duodenum were subjected to various RNA extraction protocols, and the RNA was used for quantitative RT-PCR. The subsequent use of two methods, (i) a phenol/chloroform extraction and (ii) a column-based extraction method, resulted in a yield of 4.5 microg total RNA per biopsy with reliable quality in 80% of samples. The quantitative RT-PCR analysis revealed that only RNA samples that clearly show both 18S- and 28S-RNA bands in agarose gel electrophoresis were suitable for quantitative RT-PCR as shown by expression of corpus-specific pepsinogen C-mRNA and the duodenum-specific multi-drug resistance protein-1 (mdr-1)-mRNA. In partially degraded RNA, pepsinogen C, mdr-1, or beta-actin mRNAs were still detectable, but the quantitative determination gave inconsistent data. The two-step method described here is a suitable option for extracting high-quality RNA from endoscopic biopsies when other standard protocols fail.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Application of in situ ductal perfusion to facilitate isolation of high-quality RNA from mouse pancreas.

A technique to isolate high-quality intact RNA from murine pancreas is described. This technique involves in situ ductal perfusion of the pancreas with an RNase inhibitor prior to removal of the organ for RNA extraction. In this way, the pancreatic RNases are inhibited in situ allowing good yields of intact RNA, suitable for studies on pancreatic gene transcription by real-time PCR or microarray analysis, to be obtained in a reliable way.

Animals↗
PubMed2002

High-quality RNA from cells isolated by laser capture microdissection.

Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNAfrom these cells can be problematic; the sample may deteriorate before or during sectioning, RNA may degrade during slide staining and LCM, and inadequate extraction and isolation methods may lead to poor recovery. Our report describes an optimized protocol for preparation of frozen sections for LCM using the HistoGene Frozen Section Staining Kit. This slide preparation method is combined with the PicoPure RNA Isolation Kitfor extraction and isolation of RNA from low numbers of microdissected cells. The procedure is easy to perform, rapid, and reproducible. Our results show that the RNA isolated from the LCM samples prepared according to our protocol is of high quality. The RNA maintains its integrity as shown by RT-PCR detection of genes of different abundance levels and by electrophoretic analysis of ribosomal RNA. RNA obtained by this method has also been used to synthesize probes for interrogating cDNA microarray analyses to study expression levels of thousands of genes from LCM samples.

Animals↗
PubMed1994

A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.).

The isolation of biologically active RNA from cotton (Gossypium hirsutum L.) is difficult due to interference by high levels of endogenous phenolics, polysaccharides, and secondary metabolites. A modified hot borate procedure was developed to combat these cellular constituents during tissue homogenization, resulting in the quantitative recovery of RNA suitable for hybridization analysis, in vitro translation, and cDNA synthesis. The efficacy of several hot borate buffer adjuvants for the qualitative and quantitative recovery of leaf RNA was monitored by absorbance spectra, gel electrophoresis, protein, and cDNA synthesis. Of the buffer adjuvants evaluated, polyvinylpyrrolidone-40 (PVP-40) exhibited the single, most significant impact on the yield and quality of RNA isolated from cotton leaves, although inclusion of deoxycholate and/or Nonident-40 (NP-40) further enhanced the quality of the RNA. The unsurpassed qualitative and quantitative recovery of total RNA from cotton by hot borate buffer at alkaline pH, supplemented with PVP-40, deoxycholate, and/or NP-40 had also proven satisfactory for other recalcitrant plant species as well as for especially difficult tissue types.

Boric Acids↗
PubMed2004

Isolation of high-quality RNA from gymnosperm and angiosperm trees.

An improved protocol was developed for efficient and reliable extraction of high-quality total RNA and mRNA from various tissues of spruce (Picea spp.) and poplar (Populus spp.) trees, as well as other plant species. This method was specifically optimized for tissues with high content of polysaccharides, oleoresin terpenoids, and phenolic secondary metabolites, which often co-precipitate with RNA and inhibit subsequent reverse transcription. The improved protocol yielded up to 600 micrograms of total RNA per gram of tissue suitable for standard expressed sequence tags (ESTs), full-length cDNA library construction, and for microarray applications.

Chemical Fractionation↗
PubMed2005

Isolation of high spectral quality RNA using run-on gene transcription; application to gene expression profiling of human brain.

1. Gene expression studies of human brain tissues in both health and disease require the use of intact, high spectral quality messenger RNA (mRNA). Short post-mortem tissues, when adequately controlled, provide a valuable primary source of brain mRNA. 2. Typical mRNA isolation methodologies rely on total RNA extraction from whole cells or tissues. Here we report that as an alternative approach, nuclei can be isolated from the same tissues and programmed for run-on gene transcription to generate enriched mRNA fractions. 3. This novel technique represents a significant improvement over previous methods and provides high quality, high yield mRNA samples suitable for downstream applications that include cloning, sequencing and gene expression studies using DNA array technologies.

Aged↗
PubMedDate not supplied

Isolation of high-quality RNA from white spruce tissue using a three-stage purification method and subsequent cloning of a transcript from the PR-10 gene family.

Isolation of PinmIII cDNA homologues from white spruce tissues required a rigorous RNA extraction protocol developed following assessment of three previously reported conifer RNA extraction protocols. Total RNA was extracted via several purification steps designed to minimize binding of phenolics to nucleic acids and was then subjected to caesium chloride ultra-centrifugation. This procedure produced consistently high-quality, intact RNA from both needles and roots with spectrophotometric ratios of approximately 2.0 for both 260/280 nm and 260/230 nm. Total RNA was obtained from the roots of cold-hardened white spruce seedlings for cDNA library construction. More than 2 million recombinant phage particles were generated from 5 microg of a poly(A)+RNA fraction, and ca. 1.3 million cDNA particles were amplified for storage. Approximately 500,000 primary recombinant clones were screened with an heterologous PinmIII cDNA sequence yielding a unique clone, picgl, that was very similar to members of the PR10 gene family.

Cloning, Molecular↗
PubMed2005

A new yeast poly(A) polymerase complex involved in RNA quality control.

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNAMet (tRNAiMet). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNAiMet with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNAiMet by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover.

DNA, Fungal↗
PubMed2006

RNA-quality control by the exosome.

The exosome complex of 3'-->5' exonucleases is an important component of the RNA-processing machinery in eukaryotes. This complex functions in the accurate processing of nuclear RNA precursors and in the degradation of RNAs in both the nucleus and the cytoplasm. However, it has been unclear how different classes of substrate are distinguished from one another. Recent studies now provide insights into the regulation and structure of the exosome, and they reveal striking similarities between the process of RNA degradation in bacteria and eukaryotes.

Animals↗
PubMed2000

Assessment of RNA quality by semi-quantitative RT-PCR of multiple regions of a long ubiquitous mRNA.

A simple method to assess the degree of degradation present in a total RNA preparation from cells or tissues is based on the increasing probability of RNA cleavage with increasing length of an RNA molecule. Under ideal conditions, reverse transcription of a particular mRNA species with oligo-dT as the primer generates a population of cDNAs, terminating at the 5' end of the mRNA if all template RNA molecules are intact, or at the first cleavage site 5' to the polyA if some template RNAs are partially degraded. Consequently, for cellular RNA preparations with some degradation, the 5' end of an mRNA is represented in the cDNA population to a lesser extent than the 3' end of the mRNA. We describe a sensitive assay of mRNA quality that compares the relative PCR amplification of 5' and 3' regions of a long and ubiquitous mRNA following oligo-dT-primed reverse transcription.

Animals↗
PubMed2004

High-quality RNA preparation for transcript profiling of osteocytes from native human bone microdissections.

Osteocytes, the most abundant bone cell type with important roles in tissue maintenance and pathological aberrations such as observed in bone metastases, are enclosed within a highly compact, calcified extracellular matrix. This location complicates analysis in native bone, with the consequence that despite their importance their in vivo molecular physiology is only poorly understood. We have examined the possibility of isolating osteocyte RNA for transcript profiling from native, frozen bone instead of employing the formalin-fixed, paraffin-embedded, decalcified version routinely used in histology, providing chemically modified and highly disintegrated RNAs. Bone tissue was tape-assisted cryosectioned and fixed to glass slides by support of UV-flash-triggered adhesive polymerization followed by quick hematoxylin-eosin staining to generate a guidance image for microdissection. Using an UVa-nitrogen laser, matrix-enclosed osteocytes were either excised and catapulted into RNA preparation vials or freed of accompanying nonosteocyte cellular material. The influences of bone sectioning, staining, and osteocyte capturing procedures on the prepared osteocyte RNAs were analyzed and the method was optimized accordingly. The obtained osteocyte RNAs showed the expected expression pattern of marker genes (reverse transcriptase-polymerase chain reaction), and, following conversion into fluorescent-labeled cDNAs, led to transcript profiles (cDNAchips; 2600 genes) with scatter-graph geometries indicating suitability for high-confidence evaluation. With the approach described here we introduce a methodological way for the characterization of the in vivo molecular physiology of osteocytes by functional genomics.

Bone and Bones↗
PubMed2006

An efficient method for purifying high quality RNA from wheat pistils.

Many methods are available for total RNA extraction from plants, except the floral organs like wheat pistils containing high levels of polysaccharides that bind/or co-precipitate with RNA. In this protocol, a simple and effective method for extracting total RNA from small and feathery wheat pistils has been developed. Lithium chloride (LiCl) and phenol:chloroform:isoamylalcohol (PCI) were employed and the samples were ground in microcentrifuge tube using plastic pestle. A jacket of liquid nitrogen and simplified procedures were applied to ensure thorough grinding of the pistils and to minimize the samples loss. These measures substantially increased the recovery of total RNA (approximately 50%) in the extraction process. Reliable differential display by cDNA-AFLP was successfully achieved with the total RNA after DNase treatment and reverse transcription. This method is also practicable for gene expression and gene regulation studies in floral parts of other plants.

DNA, Complementary↗
PubMed1996

OCT embedded sections of pathological specimens as a source of high quality RNA for reverse transcriptase/polymerase chain reaction.

OCT embedded cryostat sections of stored pathological specimens of non-Hodgkin's lymphoma were used to provide RNA. After reverse transcription to produce cDNA, the polymerase chain reaction was performed with primers for standard and variant forms of the CD44 molecule. Using Southern transfer and hybridisation with a probe specific for exon 4 of the CD44 gene, both standard and variant forms were visualised by autoradiography. This method was shown to be applicable to other gene products by using primers specific for the abl and bcr genes. This technique permits retrospective analysis of RNA from small amounts of stored pathological samples.

Biomarkers, Tumor↗