Total RNA suitable for molecular biology analysis

Skrypina, N. A.; Timofeeva, Timofeeva A.V.; Khaspekov, George L.; Savochkina, L. P.; Beabealashvilli, Robert Sh.

doi:10.1016/s0168-1656(03)00140-8

Cited by 62 publications

(45 citation statements)

References 21 publications

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“…Of all the criteria that we evaluated, the 28S/18S rRNA ratios were the least indicative of the integrity or the quality of the RNA sample. Although it has been suggested that 28S/18S rRNA ratios should be Ն2.0 (20 ), in good-quality total RNA samples, we have seen successful cDNA and cRNA syntheses with 28S/18S rRNA ratios as low as 0.8 (Table 2). Furthermore, we have tested RNA samples isolated from mishandled bone marrow specimens that displayed different degrees of degradation, and the 28S/18S rRNA ratios were Ͼ1.5 (see Table S1 in the online Data Supplement).…”

Section: Discussionmentioning

confidence: 75%

Evaluation of Quality-Control Criteria for Microarray Gene Expression Analysis

Dumur¹,

Nasim²,

Best

et al. 2004

Clinical Chemistry

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Background: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. Methods:In the present studies we developed qualitycontrol criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A 260 /A 280 ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix ® HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. Results: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip ® system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01).

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Section: Discussionmentioning

confidence: 75%

Evaluation of Quality-Control Criteria for Microarray Gene Expression Analysis

Dumur¹,

Nasim²,

Best

et al. 2004

Clinical Chemistry

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“…The results of our experiments with artificial stains show that many samples contain enough GAPDH mRNA which is regarded as being constantly expressed throughout the menstrual cycle [10] to provide a sufficiently low threshold cycle (Fig. 4).…”

Section: Discussionmentioning

confidence: 79%

Identification of menstrual blood by real time RT-PCR: Technical improvements and the practical value of negative test results

Bauer

Patzelt

2008

Forensic Science International

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“…RT-PCR is considered a robust method to screen for RNA contents recovered from fixed material (17) or to develop methods based on determining expression of individual genes from Rnase-rich environment like GI fluids (18). We used 18S rRNA or housekeeping genes, such as beta-actin and gapdh, which are optimal for normalization in real-time quantitative RT-PCR (19 …”

Section: Resultsmentioning

confidence: 99%

Recovery of Exfoliated Cells From the Gastrointestinal Tract of Premature Infants: A New Tool to Perform “Noninvasive Biopsies?”

et al. 2007

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ABSTRACT:To gain insight into specific gene expression in the gastrointestinal (GI) tract of preterm infants, we adapted a method to isolate exfoliated epithelial cells. Gastric residual fluid aspirates (n ϭ 89) or stool samples (n ϭ 10) were collected from 96 neonates (gestational age, 24 -36 wk). Cells were characterized by microscopic observation, cytokeratin-18 immunodetection, and expression of transcripts. The human origin of cellular DNA was confirmed by amplification of specific X and Y chromosome sequences. Isolation yielded 100 -500 cells per sample for gastric aspirates (n ϭ 8) and 10 -20 cells for fecal samples (n ϭ 5). Epithelial origin was confirmed by immunodetection of cytokeratin 18. Analyses of reverse transcribed products, using two independent methods, from 15 gastric fluid and two stool samples showed that 18S-rRNA and transcripts of beta-actin, glyceraldehyde-3-phosphate dehydrogenase (gapdh), and period1 were in quantities corresponding to at least 10 cells. On 59 aspirates, we found beta-actin transcripts (all but one), cytokeratin 18 (eight positive of eight samples), SLC26-A7-1 ( I nvestigation of gene expression in the gastrointestinal (GI) tract commonly relies on the analysis of tissue biopsies. The performance of tissue biopsies, however, is unethical in human neonates. Exfoliated or sloughed cells are naturally lost by mammals in their environment and have been used as surrogates for target cells to predict the response to bioactive food components (1) or to detect biomarkers of cancer in children with inflammatory bowel disease (2) and cancer in adults (3). Yet, the feasibility of this approach has not been assessed for the exploration of neonatal GI tract.Our aims were to determine the feasibility of this approach in human infants and assess the number, quality, and lineage of cells exfoliated that can be recovered from gastric and intestinal lumen in premature infants and to evaluate whether these cells can be used to measure the expression of specific genes of interest in a "noninvasive" fashion and thus serve as surrogates for true invasive tissue biopsy specimens. The ability to use exfoliated cells instead of biopsy or autopsy material would be highly useful to evaluate disease biomarkers in therapeutics and to explore GI functional maturation (4). We have chosen to assay clock genes as well because (1) these genes are believed to be ubiquitous in somatic cells of mammals and may be modulated in the GI tract by food intake (5) and (2) we detected the expression of these genes in cells from intestinal biopsy specimens obtained in adults in earlier studies (6). To our knowledge, even though the ability of feeding to induce and synchronize the expression of these genes has been extensively studied in peripheral tissues in laboratory rodents (7), this has not been explored in human infants, whether full term or preterm. In newborn rats, a common animal model used in neonatology (8), breast-feeding was shown to alter the circadian rhythm of the clock in the liver, with the beginn...

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Total RNA suitable for molecular biology analysis

Cited by 62 publications

References 21 publications

Evaluation of Quality-Control Criteria for Microarray Gene Expression Analysis

Evaluation of Quality-Control Criteria for Microarray Gene Expression Analysis

Identification of menstrual blood by real time RT-PCR: Technical improvements and the practical value of negative test results

Recovery of Exfoliated Cells From the Gastrointestinal Tract of Premature Infants: A New Tool to Perform “Noninvasive Biopsies?”

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