The FDA approach to standardization and quality assurance of nucleic acid tests used to screen blood and plasma donations.
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Blood Donors↗
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Results for “RNA quality”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
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[The genetic diagnosis of hematopoietic malignancy by polymerase chain reaction method].
Since 1994, chimeric gene test for major-BCR/ABL, minor-BCR/ABL, AML1/MTG8, PML/RARa, DEK/CAN, CBFb/MYH11, MLL/LTG9, E2A/PBX1, TEL/AML1, and MLL/LTG4 by reverse transcription-nested-polymerase chain reaction (RT-nested-PCR) have been routinely performed in our laboratory. The frequently requested tests were those for major-BCR/ABL, minor-BCR/ABL, AML1/MTG8, PML1/RARa, and TEL/AML1 chimeric genes, being the test for major-BCR/ABL the most frequent one, accounting for more than 50% of the total orders. By the high sensitivity for minimal residual disease(MRD) detection, these methods are extremely useful for the therapeutic control of chemotherapy and bone marrow transplantation, as well as for prediction of prognosis. However, in the most cases a well-controlled monitoring could not be obtained, due to the 6-months interval between the tests, the minimal interval allowed by the insurance. The following points should be carefully observed. (1) The setting site of the primer can affect the sensitivity and the specificity of the test; (2) For the detection of chimeric genes with multiple translocation breakpoints, a genetic DNA sequencing is necessary to confirm them; (3) In patients receiving chemotherapy or bone marrow transplantation, that show extremely low blood cell counts, false-negative tests dependent on the quality of the extracted RNA and the low volume of DNA, should be avoided by careful managements. For improvements of the MRD detection tests, the quantification of the expression levels of chimeric gene and WT1 mRNA is necessary.
Hematologic Neoplasms↗
Comparative analysis of two controlled proliferation strategies regarding product quality, influence on tetracycline-regulated gene expression, and productivity.
Overexpression of the cyclin-dependent kinase inhibitor p27 and exposure to low temperature (30 degrees C) represent two strategies to establish controlled proliferation processes for production of therapeutic proteins using Chinese hamster ovary (CHO) cells. Here we analyze the effect of growth inhibition on the quality of the human model glycoprotein SEAP (secreted alkaline phosphatase) for both strategies in monoclonal CHO-derived cell lines. Separation of purified SEAP samples using two-dimensional gel electrophoresis showed that production by proliferation-controlled CHO cultures did not alter the overall integrity of the product. Further, oligosaccharide profiles were compared using HPEC-PAD analysis. No differences were detectable between SEAP profiles obtained from p27 growth-arrested and proliferating cultures. However, production at 30 degrees C led to a significant increase in the degree of sialylation, an effect that is generally considered beneficial for the in vivo efficacy of protein therapeutics. In the production context presented here, SEAP expression is controlled by the tetracycline- (tet) repressible gene regulation system. Here we show low temperature-induced upregulation of the tetracycline-dependent transactivator (tTA). This induction has been shown by Northern blot analysis to occur at the mRNA level and is independent of the promoters driving the transactivator. We also describe a novel bottleneck in productivity at low temperature found in p27 growth-arrested CHO cells cultivated at 30 degrees C.
Alkaline Phosphatase↗
The development and use of real-time PCR for the quantification of nitrifiers in activated sludge.
Chemical analytical data has long been used to monitor the performance of activated sludge plants even though the process relies on the performance of microorganisms. It is now evident that a rapid and reliable quantitative method is required, to be able to monitor the organisms responsible for nutrient transformation and their activities, allowing avenues for more efficient nutrient removal. The development of real-time or quantitative polymerase chain reaction (PCR) also known as TaqMan or 5'-nuclease assay has allowed the rapid, quantitative analysis of DNA templates, eliminating some of the variability traditionally associated with other quantitative techniques. In this study analysis of Nitrospira spp., one of the key organisms in nitrite oxidation in wastewater treatment, was used to validate real-time PCR for the their quantification in activated sludge. A probe and primer set, targeting the 16S rRNA gene of Nitrospira spp. was designed according to the constraints of the TaqMan specifications. Samples used to evaluate the method included DNA from the sludge from full-scale wastewater treatment plants and laboratory scale systems. The reproducibility, quantitative efficiency and specificity were assessed in the evaluation. It was concluded that the method is sensitive and reproducible but has some constraints on the quantitative efficiency. A survey of full-scale systems for Nitrospira spp. was carried out and the results are presented here.
Automation↗
[Identification of Chinese crude drug snake gallbladder by DNA molecular marker].
AIM: It is difficult to identify the Chinese crude drug snake gallbladder accurately by morphological and microscopical characteristics or chemical components only. In order to solve the problem, the technique based on DNA molecular marker was introduced into the authentication of snake gallbladder. METHODS: DNA templates were extracted from the membrane or the bile of snake gallbladder, and also from the muscle of the original animal Elaphe schrenckii. About 400 bp DNA fragments of 12S rRNA gene were amplified from the templates and sequenced subsequently. RESULTS: Enough amounts of DNA templates could be extracted from a bit of membrane or bile of snake gallbladder. The sequence of amplicons from the membrane, bile and muscle of the same individual were identical completely. CONCLUSION: The technique of DNA molecular marker could be used for the authentication of snake gallbladder and bile. The results indicate that the technique could be used for the identification of crude drugs from other animal secretion. DNA sequence analysis also demonstrated that the origins of commercial snake gallbladder were complicated and more efficient quality control was necessary for supervising the crude drug in the market.
Animals↗
[A prospect of genetic tests].
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Biomarkers↗
ILK as a potential marker gene to ascertain specific adenocarcinoma cell mRNA isolation from frozen prostate biopsy tissue sections.
A major technical challenge related to gene expression profiling of tissue samples is the difficulty of procuring selected cell populations from tissues that by nature are heterogeneous, such as prostate tissue. In this study we have examined the expression of integrin-linked kinase (ILK) mRNA in prostate adenocarcinoma cells versus normal prostate epithelial cells in order to determine whether ILK could be used as a reference marker gene for prostate adenocarcinoma cell mRNA isolation. Using laser microdissection (LMD) technology and real-time PCR, together with immunohistochemistry, we have analyzed ILK mRNA expression in epithelial cells isolated from frozen prostate biopsy specimens as well as 4 prostate cell lines (RWPE-1, LNCaP, PC-3 and DU 145) and correlated ILK mRNA expression with ILK protein expression. We demonstrate that quantitative upregulation of ILK mRNA expression in prostate epithelial cells derived from prostate tissue correlated with ILK protein expression and with the histopathology diagnosis of prostate adenocarcinoma. We further show that the level of ILK overexpression was directly influenced by the method used to isolate prostate adenocarcinoma cells (bulk tissue versus LMD dissected cells). These data provide evidence that ILK mRNA is quantitatively upregulated in prostate adenocarcinoma cells versus normal epithelial cells and is therefore a useful internal reference gene marker to evaluate the quality of prostate adenocarcinoma cell derived mRNA used for large scale prostate cancer cDNA gene profiling.
Adenocarcinoma↗
Quality control for microarray experiments.
OBJECTIVES: In this paper we give an overview of post-hybridization quality control methods for gene expression chips, including methods for the gene/spot level, the hybridization/chip level and the process level. We present quality control methods that can be applied after hybridization and image analysis, i.e. that use data from the chip experiment itself. Wet lab quality control steps, which should be applied before the probe is measured on a chip, are not discussed. This review is aimed towards statisticians and data analysts. METHODS: We give examples of some of the quality control measures available for spotted cDNA and Affymetrix GeneChips, the most common chip types. As quality control measures are technology and design-dependent, we will stress on methods that have the potential to be applied platform-independently. RESULTS: Quality control should identify poor quality chips or hybridizations, as well as faulty measurements for individual genes/spots. Additionally, high throughput laboratories processing several tens or hundreds of microarrays per week have the need for an appropriate process control to be able to identify changes in the production process as early as possible. CONCLUSION: Microarrays have become a standard research tool for biologists and medical researchers. As a consequence, there is a great need for standardized quality control, as false findings due to problem in data quality can lead to a substantial loss of resources.
DNA Probes↗
Avoid DNA/RNA contamination in polymerase chain reactions.
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DNA↗
A cautionary note on reaction tubes for differential display and cDNA amplification in thermal cycling.
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DNA, Complementary↗
Virus isolation accuracy: an evaluation of eight laboratories involved in poultry diagnostics.
A study was undertaken to evaluate the accuracy of diagnostic laboratories at isolation of common avian viral pathogens. Fourteen "unknown" samples were submitted to eight laboratories in seven states. All positive and negative samples were guaranteed pure by SPAFAS. Virus-isolation results were erroneous in many cases. Based on this study, it appears that protocol for virus isolation of avian pathogens should be standardized throughout the United States.
Animals↗
Synergistic enhancement of both initiation and elongation by acidic transcription activation domains.
The effects of activation domain synergy on transcription initiation and elongation have been examined utilizing a system that permits the targeting of a defined number of activation modules to promoter DNA. As predicted, incremental increases in targeted activation potential were found to result in corresponding increases in transcription initiation. Surprisingly, however, transcriptional processivity, and hence mRNA synthesis, required a threshold level of activation domain synergy that exceeded the level required for at least modest levels of transcription initiation. The degree to which transcriptional processivity was enhanced was shown to depend on the quantity of activation modules targeted to the promoter DNA, rather than the quality. While the RNA-sequence specific HIV-1 Tat trans-activator was also shown to enhance processivity in this assay system, Tat differed from DNA-sequence specific activation domains in exerting a more dramatic effect on the efficiency of transcript elongation.
Animals↗
Gene-expression analysis of single cells-nested polymerase chain reaction after laser microdissection.
AIM: The structural and functional characteristics of cells are dependent on the specific gene expression profile. The ability to study and compare gene expression at the cellular level will therefore provide valuable insights into cell physiology and pathophysiology. METHODS: Individual cells were isolated from frozen colon tissue sections using laser microdissection. DNA as well as RNA were extracted, and total RNA was reversely transcribed to complementary DNA (cDNA). Both DNA and cDNA were analyzed by nested polymerase chain reaction (PCR). The quality of isolated DNA and RNA was satisfactory. RESULTS: Single cells were successfully microdissected using an ultraviolet laser micromanipulator. Nested PCR amplification products of DNA and cDNA of single cells could clearly be visualized by agarose gel electrophoresis. CONCLUSION: The combined use of laser microdissection and nested-PCR provides an opportunity to analyze gene expression in single cells. This method allows the analysis and identification of specific genes which are involved in physiological and pathophysiological processes in a complex of variable cell phenotypes.
Colon↗
Validation of cDNA microarray gene expression data obtained from linearly amplified RNA.
BACKGROUND: DNA microarray technology has permitted the analysis of global gene expression profiles for several diseases, including cancer. However, standard hybridisation and detection protocols require micrograms of mRNA for microarray analysis, limiting broader application of this technology to small excisional biopsies, needle biopsies, and/or microdissected tissue samples. Therefore, linear amplification protocols to increase the amount of RNA have been developed. The correlation between the results of microarray experiments derived from non-amplified RNA and amplified samples needs to be evaluated in detail. METHODS: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (aRNA) and non-amplified mRNA from tonsillar B cells and the SUDHL-6 cell line using cDNA microarrays containing approximately 4500 genes. The results of microarray differential expression using either source of RNA (mRNA or aRNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: Microarray experiments using aRNA generated reproducible data displaying only small differences to data obtained from non-amplified mRNA. The quality of the starting total RNA template and the concentration of the promoter primer used to synthesise cDNA were crucial components of the linear amplification reaction. Approximately 80% of selected upregulated and downregulated genes identified by microarray analysis using linearly amplified RNA were confirmed by QRT-PCR using non-amplified mRNA as the starting template. CONCLUSIONS: Linear RNA amplification methods can be used to generate high fidelity microarray expression data of comparable quality to data generated by microarray methods that use non-amplified mRNA samples.
Gene Amplification↗
On precision limits for derivatives numerically calculated from noisy data.
This paper has three purposes. (1) To verify an error formula from which the maximal precision in derivatives obtained from noisy measurement data can be calculated. The formula is verified by comparison with the resulting noise in derivatives obtained by local least squares polynomial fitting. It is also verified that differentiation by Fourier series expansion gives noise exceeding the minimum value according to the error formula. (2) An index called the "Relative Noise Amplification" (RNA) is introduced. For an arbitrary differentiating filter it is defined as the noise transmission of the filter divided by the minimal noise transmission according to the above mentioned error formula. When the filter produces unbiased estimates the value of the RNA always exceeds one. The RNA can be used as a quality index for differentiating filters. The filter with the smallest value on the RNA also has the smallest amount of noise superimposed on the calculated derivatives provided the input noise is white and additive. (3) The bandwidth and the Relative Noise Amplification of differentiating filters obtained by local least squares polynomial fitting are presented. The 0th, 1st and 2nd order derivatives have been investigated for polynomials up to 8th order. These results can be used for the determination of suitable filter parameters in various applications.
Biomechanical Phenomena↗
Isolation of erythroid cells from the mouse embryonic yolk sac by laser capture microdissection and subsequent microarray hybridization.
Erythropoietic tissues are complex, containing both erythroid and other cells. The embryonic yolk sac in particular contains primitive erythroid cells in low abundance. Laser capture microdissection (LCM) was performed to isolate erythroid cells, and epithelial cells, from mouse embryonic day 10 (E10) yolk sac. Quantitative RT-PCR was performed to confirm that enriched cell populations were obtained. epsilony- and betaH1-globin mRNAs were enriched in the erythroid compared to the epithelial fraction, and villin mRNA was enriched in the epithelial compared to the erythroid fraction. RNA isolated from the microdissected erythroid cells was of high quality as indicated by capillary electrophoresis. The RNA from the LCM erythroid fraction was linearly amplified with T7 RNA polymerase and hybridized to a Mouse 430A 2.0 Affymetrix array. Forty-eight percent of genes were present in the microarray assays, including low abundance transcripts such as erythroid transcription factors and enzymes involved in heme synthesis. With the LCM/microarray strategy, it will be possible to identify genes that are differentially regulated in native primitive and definitive erythroid cells.
Animals↗
HIV protease inhibitor substitution in patients with lipodystrophy: a randomized, controlled, open-label, multicentre study.
BACKGROUND: Lipodystrophy, dyslipidaemia and insulin resistance often complicate protease inhibitor-containing antiretroviral therapy. The aims of this study were to determine if these are reversible with continued HIV suppression following protease inhibitor substitution. METHODS: Eighty-one HIV protease inhibitor recipients (78 male; mean antiretroviral therapy, 55 months) with predominant peripheral lipoatrophy, HIV RNA < 400 copies/ml plasma for at least the preceding 6 months, and no prior abacavir, non-nucleoside analogue or adefovir therapy were randomized 3 : 2 to continue nucleoside analogues and substitute protease inhibitor(s) with abacavir, nevirapine, adefovir and hydroxyurea (n = 49) or to continue all therapy (n = 32) with an option to switch at week 24. The primary endpoints were total body fat and HIV RNA at week 24. Other assessments were regimen safety, regional body composition, metabolic parameters, quality of life, and CD4 T-lymphocyte counts to week 48. RESULTS: There was a greater decline in total body fat in the switch group than in the continue group (-1.6 and -0.4 kg, respectively at week 24; P = 0.006). This comprised greater declines in limb and subcutaneous abdominal fat, and in intra-abdominal fat of patients with moderate or severe abdominal fat accumulation. Viral suppression was similar, despite 18 (37%) switch group patients ceasing at least one study drug by week 24 because of adverse events. Total cholesterol and triglycerides declined more in the switch group (both P < 0.002). High density lipoprotein cholesterol increased significantly in both groups at week 48 (P < 0.02). There was no change for any glycaemic parameter. CONCLUSIONS: In predominantly lipoatrophic patients, switching from HIV protease inhibitor therapy lead to improved lipids and less intra-abdominal fat, but also to less peripheral fat, and had minimal effect on insulin resistance. Virological control in these heavily pretreated patients was unaffected, despite frequent switch drug cessations.
Adenine↗
Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world affecting the oral cavity, salivary glands, larynx and pharynx. Utilizing tissue from patients with HNSCC, we sought to systematically identify and catalog genes expressed in HNSCC progression. Here, we demonstrate the successful use of laser capture microdissection for procuring pure populations of cells from patient tissue sets comprised of oral squamous cell carcinomas (OSCCs) and matching normal tissue. From the estimated 5000 cells procured for each sample, we were able to extract total RNA (14.7-18.6 ng) of sufficient quality to transcribe GAPDH by reverse transcriptase-polymerase chain reaction (RT-PCR). The RNA was used for the synthesis of blunt-ended, double-strand complementary DNAs (cDNAs) by oligo (dT)-mediated reverse transcription, followed by addition of linkers. Primers specific for these linkers with uracil deglycosylase-compatible ends were used to amplify these cDNAs by PCR and the product was subcloned into the pAMP10 cloning vector. Ninety-six clones from each of six libraries were randomly sequenced and results indicated that 76-96% of the inserts represent either anonymous expressed sequence tags (ESTs) (25-48%), known genes (9-29%) or novel sequences (27-51%), respectively, with very little redundancy. These results demonstrate that high quality, representative cDNA libraries can be generated from microdissected OSCC tissue. Furthermore, these finding suggest the existence of at least 132 novel genes expressed in our cDNA libraries, which may have a role in the pathogenesis of HNSCC, and may represent novel markers for early detection as well as targets for pharmacological intervention in this disease.
Aged↗