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PubMed2002

Laser capture microdissection of Plasmodium falciparum liver stages for mRNA analysis.

Plasmodium falciparum liver-stages are important targets for vaccine-induced protective immune responses and prophylactic treatment against malaria. Little is known of the gene expression profile of malaria parasites during their development inside hepatocytes. The sequencing of the P. falciparum genome and the development of DNA microarray technology give new opportunities to identify genes expressed during the development of Plasmodium. However, transcriptome analysis cannot currently be applied to the hepatic stages, due to difficulties in obtaining sufficient amounts of parasite material that lie among the large excess of host cell RNA. Here, we describe the isolation of liver-stages by a modified laser capture microdissection approach applied to human hepatocyte cultures infected with P. falciparum. RT-PCR amplification of several P. falciparum transcripts demonstrated the high quality of the RNA recovered after microdissection. This approach should enable analyses of P. falciparum transcriptome during its hepatic development and substantially assist the identification of new therapeutic and vaccine targets.

Animals↗

PubMed2005

Surveillance of transcriptomes in basic military trainees with normal, febrile respiratory illness, and convalescent phenotypes.

Gene expression profiles permit analysis of host immune response at the transcriptome level. We used the Pax gene Blood RNA (PAX) System and Affymetrix microarrays (HG-U133A&B) to survey profiles in basic military trainees and to classify them as healthy, febrile respiratory illness (FRI) without adenovirus, FRI with adenovirus, and convalescent from FRI with adenovirus. We assessed quality metrics of RNA processing for microarrays. Class prediction analysis discovered nested sets of transcripts that could categorize the phenotypes with optimized accuracy of 99% (nonfebrile vs febrile, P<0.0005), 87% (healthy vs convalescent, P=0.001), and 91% (febrile without vs with adenovirus, P<0.0005). The discovered set for classification of nonfebrile vs febrile patients consisted of 40 transcripts with functions related to interferon induced genes, complement cascades, and TNF and IL1 signaling. The set of seven transcripts for distinguishing healthy vs convalescent individuals included those associated with ribosomal structure, humoral immunity, and cell adhesion. The set of 10 transcripts for distinguishing FRI without vs with adenovirus had functions related to interferon induced genes, IL1 receptor accessory protein, and cell interactions. These results are the first in vivo demonstration of classification of infectious diseases via host signature transcripts and move us towards using the transcriptome in bio-surveillance.

Adenovirus Infections, Human↗

PubMed2003

Optimization of RNA yield, purity and mRNA copy number by treatment of urine cell pellets with RNAlater.

BACKGROUND: We have shown that measurement of mRNA for cytotoxic attack proteins perforin and granzyme B in urinary cells is a noninvasive means of diagnosing acute rejection of human renal allografts. Urinary cell mRNA studies have yielded useful information in other patient populations such as patients with cancer. The isolation of sufficient and high quality ribonucleic acid (RNA) from urinary cells however is problematic. RNAlater, an RNA stabilization solution, has been reported to optimize RNA isolation from tumor tissues stored at room temperature and from pigment-rich ocular tissues. METHODS: We explored whether the addition of RNAlater to urine cell pellets improves RNA yield, enhances purity and facilitates measurement of low abundance mRNAs. We measured, with the use of real-time quantitative polymerase chain reaction (PCR) assay, levels of expression of a constitutively expressed gene 18S rRNA and mRNA for granzyme B and transforming growth factor-beta(1) (TGF-beta(1)) in urine specimens and renal biopsies obtained from renal allograft recipients. RESULTS: RNA yield (P<0.01, Wilcoxon signed rank test) and the A260/A280 ratio (P<0.01) were both higher with urine cell pellets treated with RNAlater prior to snap freezing compared to cell pellets that were not treated with RNAlater prior to snap freezing. Levels (copy number per 1 microg of total RNA) of 18S rRNA (P<0.02), granzyme B mRNA (P=0.002) and TGF-beta(1) (P=0.02) were all higher with treated urine cell pellets compared to untreated cell pellets. Kruskall-Wallis one way analysis of variance and pair-wise comparisons with Student-Newman-Keuls test showed that the levels of mRNA for granzyme B (P<0.05) and TGF-beta(1) (P<0.05) are significantly different between renal allograft biopsies and untreated urine cell pellets but not between the biopsy specimens and RNAlater-treated urine cell pellets. CONCLUSIONS: The addition of RNAlater to urine cell pellets improves RNA isolation from urinary cells and facilitates measurement of low abundance mRNAs.

Adolescent↗

PubMed2006

Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA.

Reverse transcription of RNA is an invaluable method for gene expression analysis by real-time PCR or microarray methods. Random primers of varying lengths were compared with respect to their efficiency of priming reverse transcription reactions. The results showed that 15-nucleotide-long random oligonucleotides (pentadecamers) consistently yielded at least 2-fold as much cDNA as did random hexamers using either poly(A) RNA or an amplified version of messenger RNA (aRNA) as a template. The cDNA generated using pentadecamers did not differ in size distribution or the amount of incorporated label compared with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of >80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage of the transcriptome when using random pentadecamers over random hexamers. Using the same amount of aRNA as starting material, random pentadecamer-primed reactions resulted in 11-fold more genes being detected in whole transcriptome DNA microarray experiments than random hexamer-primed reactions. The results indicate that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality.

DNA Primers↗

PubMedDate not supplied

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

Actins↗

PubMed1999

The contribution of assay variation and biological variation to the total variability of plasma HIV-1 RNA measurements. The Women Infant Transmission Study Clinics. Virology Quality Assurance Program.

OBJECTIVES: To assess the specific contributions of assay variation and biological variation to the total variation of plasma HIV-1 RNA measured by the Roche Monitor assay and the extent to which batch assays reduced both assay variability and total variability compared with real-time determinations. DESIGN: A retrospective analysis of data obtained from three trials conducted by the Adult and Pediatric AIDS Clinical Trials Groups (ATCG), the Women and Infants Transmission Study (WITS) and the NIAID-sponsored Virology Quality Assurance Program. METHODS: Within-subject variation was assessed from stored, serially collected plasma samples from 663 subjects enrolled in the ACTG and WITS studies. Interassay and intra-assay variation were estimated from two of the clinical trials and 22 laboratories that participated in a quality assurance program and were used to estimate the effect of real-time testing on total variation. RESULTS: The total variation (standard deviation) from a random effects model was 0.26 log10 RNA copies/ml. The estimated interassay variation was 0.08 log10 and intra-assay variation was 0.12 log10 RNA copies/ml. Biological variation accounted for 56-80% of total variation. The effect of real-time testing compared with batch testing was minimal. CONCLUSION: Our estimates of total within-subject HIV-1 RNA variation support the current recommendation to obtain at least two specimens, preferably obtained less than 2 weeks apart, for viral RNA measurement before starting therapy. The major contribution of biological variation to the total variation supports the use of real-time HIV-1 RNA assays, provided that consistent specimen collection procedures are followed and acceptable assay proficiency is maintained.

Adult↗

PubMed2006

Same-sample analysis of ethoxyresorufin-O-deethylase activity and cytochrome P4501A mRNA abundance in chicken embryo hepatocytes.

Inducibility of the cytochrome P4501A4 (CYP1A4) enzyme, measured as ethoxyresorufin-O-deethylase (EROD) activity, has been used as a biomarker for sensitivity to the effects of dioxin-like compounds in avian species. Here, we present a quantitative reverse transcriptase-PCR (Q-PCR) method for assessing this biomarker response at the level of messenger RNA (mRNA) expression. The method was validated for use in fresh samples as well as samples that have been analyzed for EROD activity previously. Concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on CYP1A4 and CYP1A5 mRNA abundance were detected in fresh and post-EROD hepatocyte cultures. Although the quality of the RNA obtained from post-EROD samples was low, quantification of the CYP1A mRNA response to TCDD was not compromised. Several benefits of evaluating CYP1A mRNA expression in addition to EROD activity were noted. The CYP1A mRNA bioassay may provide more accurate estimates for the potency of environmental mixtures of contaminants and has a very low detection limit. When working with hepatocytes cultured from wild or endangered species, our approach can help to circumvent the problem of small sample size by maximizing the amount of data obtained from each sample.

Animals↗

PubMed1991

A comparison of 18s ribosomal RNA and rubisco large subunit sequences for studying angiosperm phylogeny.

Partial sequences of 18s rRNA were obtained for 2 gymnosperms and 12 angiosperms from a wide range of families and these were analyzed with 5 other published sequences to form a phylogenetic tree. Using 16 published sequences of the large subunit of rubisco (rbcL), also from a wide range of angiosperm families, another phylogenetic tree was derived and the two approaches were compared. Both phylogenetic trees gave good grouping within families but in neither case was there resolution of the branching order of major taxa. Superficially the long rbcL sequences (whose base composition was homogeneous among all species) seemed very promising, but analysis showed that a large proportion of the variation did not affect the amino acid sequence. Although silent substitution contained some phylogenetic information, at the level required to order major taxa, much of it was random and obfuscating. It was concluded that neither macromolecule alone was likely to yield a solution to the problem of angiosperm phylogeny and therefore that studies of both, at least, will be required. For this reason, a method was described for obtaining both DNA and RNA of good quality from the same preparation and which had been used successfully with a wide range of species including many with pungent leaves.

Base Sequence↗

PubMed1991

Selection of small molecules by the Tetrahymena catalytic center.

The catalytic center in group I RNAs contains a selective binding site that accommodates both guanosine and L-arginine. In order to understand the specificity of the RNA for small molecules, we analyzed 6 RNAs that vary in this region. Specificity for nucleotides resides substantially in G264 rather than its paired nucleotide C311, and is expressed substantially in Km, with comparatively little variation in kcat. kcat is not notably perturbed even for RNAs with mispairs in the active-site helix. For 5 of 6 sequences, effects of RNA substitutions on arginine binding and GTP reactivity are proportional, confirming that arginine contacts a subset of the groups occupied by G. As a result of particular mutations, reaction with GTP is decreased, and reaction with the natural nucleotides UTP and ATP is enhanced. Molecular modeling of these effects suggests that exceptionally flexible placement of reactants may be an essential quality of RNA-catalyzed splicing. The specificity of the intron can be rationalized by a type of binding model not previously considered, in which the G/arginine site includes adjacent nucleotides (an 'axial' site), rather than a single nucleotide, G264.

Amino Acids↗

PubMed2000

Isolation and purification of functional total RNA from woody branches and needles of Sitka and white spruce.

The isolation of intact, functional RNA from conifer spp. is not easy, especially from those tissues that are heavily lignified and characterized by a low number of living cells. An efficient procedure for isolating RNA from combined wood and bark tissues of conifers was developed based on a protocol optimized for the extraction of RNA from pollen and one for the isolation of RNA from woody stems. This protocol does not involve the use of phenol, and no ultracentrifugation was required. In addition, the protocol overcame the problems of RNA degradation and low yield due to oxidation by polyphenolics and co-precipitation with polysaccharides, both of which are abundant components in conifer bark tissues. The isolated RNA was of high quality and undegraded as gauged by spectrophotometric readings and electrophoresis in denaturing agarose gels. Quality was further assessed through the subsequent use of the RNA in reverse transcription and RT-PCR, indicating that it could be used for a number of downstream purposes including Northern blot hybridization and cDNA library construction. Using this modified protocol, 80-150 micrograms of RNA was routinely obtained from 1 g of fresh material. This protocol was also used for the isolation of RNA from needles of spruce spp., from which 750-950 micrograms RNA per gram of starting material could routinely be obtained.

Base Sequence↗

PubMed2003

Feasibility of using low-volume tissue samples for gene expression profiling of advanced non-small cell lung cancers.

PURPOSE: The majority of patients with non-small cell lung cancer (NSCLC) present at an advanced clinical stage, when surgery is not a recommended therapeutic option. In such cases, tissues for molecular research are usually limited to the low-volume samples obtained at the time of diagnosis, usually via fine-needle aspiration (FNA). We tested the feasibility of performing gene expression profiling of advanced NSCLCs using amplified RNA from lung FNAs. EXPERIMENTAL DESIGN AND RESULTS: A total of 46 FNAs was tested, of which 18 yielded RNA of sufficient quality for microarray analysis. Expression profiles of these 18 samples were compared with profiles of 17 pairs of tumor and normal lung tissues that had been surgically obtained. Using a variety of unsupervised and supervised analytical approaches, we found that the FNA profiles were highly distinct from the normal samples and similar to the tumor profiles. CONCLUSIONS: We conclude that when RNA amplification is successful, gene expression profiles from NSCLC FNAs can determine malignancy and suggest that with additional refinement and standardization of sample collection and RNA amplification protocols, it will be possible to conduct additional and more detailed molecular analysis of advanced NSCLC using lung FNAs.

Biopsy, Needle↗

PubMed2002

Analysis of the molecular quality of human tissues: an experience from the Cooperative Human Tissue Network.

The scientific usefulness of the data obtained from tissue analysis is related to specimen quality, which may be affected by conditions that may contribute to the degradation of the specimen before processing and analysis. We determined the usability of nucleic acids extracted from banked human tissues for further molecular analyses. We assayed 151 tissue specimens, storedfor various times at 4 divisions of the Cooperative Human Tissue Network, National Cancer Institute, Bethesda, MD, for DNA and RNA degradation. Simple electrophoresis, polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, and Northern blot analysis were compared to determine the optimal quality control procedure. In addition, a time course degradation procedure was performed on human lung tissue. Gel electrophoresis was as informative as PCR, RT-PCR, and Northern blot analysis in determining the molecular usefulness of the human tissues. Overall, 80% of the stored human tissues had good-quality DNA, and 60% had good-quality RNA. Electrophoresis procedures for DNA and RNA offer a quick and valuable measure of the molecular quality of stored human tissues. The DNA and RNA degradation of one tissue type (lung) was stable for both nucleic acids for up to 5 hours after excision.

Blotting, Northern↗

PubMed2003

Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA.

In this paper we show that RNase R is a cold shock protein that is induced seven- to eightfold by cold shock and that its expression is tightly regulated by temperature. Transcriptional studies reveal that the rnr gene is co-transcribed with flanking genes as an operon induced under cold shock. The induction of RNase R levels is mainly a result of the stabilization of the rnr transcripts. The transient stability of the rnr transcripts is shown to be regulated by PNPase at the end of the acclimation phase. Studies with an rnr mutant revealed a cold-shock phenotype showing that RNase R contributes to growth at low temperatures. We have shown that RNase R can be involved in the maturation of SsrA/tmRNA, an important small stable RNA involved in protein tagging and ribosome rescue. The wide biological significance of RNase R regarding adaptation to cold shock and its involvement in RNA surveillance, protein quality control and pathogenesis is discussed.

Bacterial Proteins↗

PubMed2006

Isolation of RNA and DNA from leukocytes and cDNA synthesis.

In this chapter, methods to isolate RNA and DNA from human leukocytes for the subsequent use in molecular diagnostic tests are described. In addition, protocols for cDNA synthesis are given, both for the use in conventional reverse transcription (RT)-polymerase chain reaction (PCR), and for the use in quantitative RT-PCR reactions. Because sensitive PCR methods are commonly used in molecular diagnostics, measures to avoid contamination are described. Finally, a simple procedure to control the quality of isolated RNA is described.

DNA↗

PubMed2003

Structured treatment interruption in patients with multidrug-resistant human immunodeficiency virus.

BACKGROUND: We compared two strategies for treating patients infected with multidrug-resistant human immunodeficiency virus (HIV). METHODS: Patients with multidrug-resistant HIV and HIV RNA levels of more than 5000 copies per milliliter were randomly assigned to a four-month structured interruption of treatment followed by a change in antiretroviral regimen (treatment-interruption group) or to an immediate change in regimen (control group). Genotypic and phenotypic resistance testing was performed. Disease progression, death, and changes in genotypic resistance, CD4 cell counts, HIV RNA levels, and quality of life were assessed. RESULTS: After a median follow-up of 11.6 months, disease progression or death occurred in 22 of the 138 patients in the treatment-interruption group and in 12 of the 132 patients in the control group (P=0.01), with a hazard ratio of 2.57 (95 percent confidence interval, 1.2 to 5.5) for the treatment-interruption group. There were eight deaths in each group. In the treatment-interruption group, the mutant HIV populations completely or partially reverted to wild type by four months in 64.0 percent of patients. As compared with the control group, the treatment-interruption group had a mean CD4 cell count that was 85 cells per cubic millimeter lower from months 0 through 4 (P<0.001), 47 cells per cubic millimeter lower from months 5 through 8 (P<0.001), and 31 cells per cubic millimeter lower after eight months (P=0.11). The mean HIV RNA levels were 1.2 log copies per milliliter higher (on a base-10 scale) in the treatment-interruption group during months 0 through 4 (P<0.001), but they were not significantly different from those in the control group after month 4. The overall quality of life was similar in the two groups. CONCLUSIONS: In patients infected with multidrug-resistant HIV, structured interruption of treatment was associated with greater progression of disease and did not confer immunologic or virologic benefits or improve the overall quality of life.

AIDS-Related Opportunistic Infections↗

PubMed1997

RAGA: RNA sequence alignment by genetic algorithm.

We describe a new approach for accurately aligning two homologous RNA sequences when the secondary structure of one of them is known. To do so we developed two software packages, called RAGA and PRAGA, which use a genetic algorithm approach to optimize the alignments. RAGA is mainly an extension of SAGA, an earlier package for multiple protein sequence alignment. In PRAGA several genetic algorithms run in parallel and exchange individual solutions. This method allows us to optimize an objective function that describes the quality of a RNA pairwise alignment, taking into account both primary and secondary structure, including pseudoknots. We report results obtained using PRAGA on nine test cases of pairs of eukaryotic small subunit rRNA sequence (nuclear and mitochondrial).

Algorithms↗

PubMed1993

Biological variation and quality control of plasma human immunodeficiency virus type 1 RNA quantitation by reverse transcriptase polymerase chain reaction.

Quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in the plasma of seropositive individuals was performed by using an external control assay with techniques to standardize and control each measurement. Rigorous study of the variability of the assay showed that the median intraassay reproducibility was log10 0.15 RNA copies per ml of plasma, while the median interassay reproducibility on replicate plasma samples was log10 0.25 copies perml. Specimen stability studies showed reproducible recovery of RNA from plasma stored at -70 degrees C for up to 12 months. In clinically stable patients who were either untreated or taking zidovudine, the average week-to-week variation in plasma RNA levels, measured in real time, was log10 0.30 RNA copies per ml. In contrast, patients either initiating or changing antiretroviral therapy showed a fall of log10 0.8 to log10 2.0 copies per ml in plasma RNA levels. Overall, 105 of 110 (96%) HIV-1-seropositive individuals with CD4 counts of 36 to 868 cells per mm3 had quantifiable HIV-1 RNA over a range of log10 2.70 to log10 6.23 RNA copies per ml, including 81% (13 of 16) of the individuals with greater than 500 CD4 cells per mm3. Accurate and reproducible quantitation of plasma viremia in real time by reverse transcriptase polymerase chain reaction, particularly in asymptomatic HIV-1-infected individuals with high CD4 counts, provides a basis for the use of this virologic measure to monitor the short- and long-term effects of early intervention therapeutic strategies on viral burden.

CD4-Positive T-Lymphocytes↗

PubMed2006

The subcellular localisation of trypanosome RRP6 and its association with the exosome.

The exosome, a complex of 3'-exoribonucleases and associated proteins, is involved in the degradation of eukaryotic mRNAs in the cytoplasm, and has RNA processing and quality control functions in the nucleus. In yeast, the nuclear exosome differs from the cytoplasmic one in that it contains an additional non-essential component, Rrp6p. In contrast, a small proportion of human RRP6 has been shown to localise to the cytoplasm as well. When we purified the Trypanosoma brucei exosome from cytosolic extracts we found RRP6, apparently in stoichiometric amounts. We here confirm that RRP6 is in the trypanosome cytoplasm and nucleus. The level of RRP6 was unaffected by depletion of core exosome subunits by RNA interference and over-expression of tagged RRP6 was possible, indicating that RRP6 can be present independent of exosome association.

Animals↗