Method validation of <b><i>in vitro</i></b> RNA transcript analysis on the Agilent 2100 Bioanalyzer

Sodowich, Bruce I.; Fadl, Irini; Burns, Craig

doi:10.1002/elps.200600673

Cited by 23 publications

(15 citation statements)

References 29 publications

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“…The RNA integrity assays (32) were performed using a 2100 Bioanalyzer (Agilent Techologies, Santa Clara, CA) with an RNA 6000 Pico kit and Ladder (Ambion/Applied Biosystems, Austin, TX), according to the manufacturer’s instructions. The bioanalyzer produces an electropherogram, which shows the distribution of RNA transcripts in the sample.…”

Section: Methodsmentioning

confidence: 99%

Metabolic, pathologic, and genetic analysis of prostate tissues: quantitative evaluation of histopathologic and mRNA integrity after HR-MAS spectroscopy

et al. 2009

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The impact of high-resolution magic angle spinning (HR-MAS) spectroscopy on the histopathologic and mRNA integrity of human prostate tissues was evaluated. Forty prostate tissues were harvested at transrectal ultrasound (TRUS) guided biopsy (n =20) or radical prostatectomy surgery (n =20), snap-frozen on dry ice, and stored at −80°C until use. Twenty-one samples (n =11 biopsy, n =10 surgical) underwent HR-MAS spectroscopy prior to histopathologic and cDNA microarray analysis, while 19 control samples (n =9 biopsy, n =10 surgical) underwent only histopathologic and microarray analysis. Frozen tissues were sectioned at 14-μm intervals and placed on individual histopathology slides. Every 8th slide was stained with hematoxylin and eosin (H&E) and used to target areas of predominantly epithelial tissue on the remaining slides for mRNA integrity and cDNA microarray analysis. Histopathologic integrity was graded from 1 (best) to 5 (worst) by two 'blinded' pathologists. Histopathologic integrity scores were not significantly different for post-surgical tissues (HR-MAS vs controls); however, one pathologist's scores were significantly lower for biopsy tissues following HR-MAS while the other pathologist's scores were not. mRNA integrity assays were performed using an Agilent 2100 Bioanalyzer and the electrophoretic traces were scored with an RNA integrity number (RIN) from 1 (degraded) to 10 (intact). RIN scores were not significantly different for surgical tissues, but were significantly lower for biopsy tissues following HR-MAS spectroscopy. The isolated mRNA then underwent two rounds of amplification, conversion to cDNA, coupling to Cy3 and Cy5 dyes, microarray hybridization, imaging, and analysis. Significance analysis of microarrays (SAM) identified no significantly over-or under-expressed genes, including 14 housekeeping genes, between HR-MAS and control samples of surgical and biopsy tissues (5% false discovery rate). This study demonstrates that histopathologic and genetic microarray analysis can be successfully performed on prostate surgical and biopsy tissues following HR-MAS analysis; however, biopsy tissues are more fragile than surgical tissues.

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Section: Methodsmentioning

confidence: 99%

Metabolic, pathologic, and genetic analysis of prostate tissues: quantitative evaluation of histopathologic and mRNA integrity after HR-MAS spectroscopy

et al. 2009

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“…The concentration (ng/μL) of freshly extracted total RNA was quantitated using a UV/visible spectrophotometer (UItrospec 4300 pro: Biochrom, Cambridge, UK). RNA integrity was assessed using an Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA) by loading samples onto the Eukaryote total RNA nano-chip [12,13].…”

Section: Total Rna Preparationmentioning

confidence: 99%

Time-dependent expression of skeletal muscle troponin I mRNA in the contused skeletal muscle of rats: a possible marker for wound age estimation

et al. 2009

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To estimate the age of skeletal muscle contusion, the expression of troponin I mRNA in contused skeletal muscle of rats was detected using real-time polymerase chain reaction (PCR). A total of 51 Sprague-Dawley male rats were divided into control and contusion groups, and another nine rats received contusion injury after death. At 0.5, 1, 6, 12, 18, 24, 30, and 36 h after contusion, the rats were killed with a lethal dose of pentobarbital. Total RNA was isolated from muscle specimens using the SV Total RNA Isolation System and reverse transcribed into first-strand cDNA. Sequence-specific primers were then used to conduct real-time PCR to analyze the expression levels of sTnI mRNA. At 0.5, 1, and 6 h after contusion, the expression levels of sTnI mRNA decreased to 78.17% (P < 0.05), 41.58% (P < 0.05), and 32.13% of that in the control group, respectively. However, there were no significant changes in the expression levels of sTnI mRNA from 6 to 36 h (P > 0.05) after contusion when normalized to RpL32 expression. The expression levels of sTnI mRNA in the normal and contused skeletal muscle of postmortem rats were about 70% of that in the control group (P < 0.05), and no significant changes in the expression levels of sTnI mRNA in the postmortem contusion group were noted among different time points after injury. This result suggests that determination of sTnI mRNA levels by real-time PCR is useful for the estimation of wound age.

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“…The in vitro transcribed RNAs were cleaned up using an RNeasy 1 kit (Qiagen) according to the manufacturer's instructions. All RNA concentrations were measured with a 2100 Bioanalyzer (Agilent, Palo Alto, CA) (Sodowich et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

Sensitive multiplex RNA quantification using capillary electrophoresis‐based single‐strand conformation polymorphism

Shin¹,

Hwang²,

Nam³

et al. 2010

Biotech & Bioengineering

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Quantification of RNA provides information crucial for various biological studies, including analysis of mRNA expression and that of microRNAs. Reverse transcription (RT) coupled with real-time polymerase chain reaction (PCR) is known to be the most accurate method for quantifying nucleic acids, and thus represents the state-of-the-art for RNA quantification. However, the use of real-time PCR for RNA quantification is limited to a single target per analytical run because of reductions in quantification power and limitations of fluorescence dyes associated with multiplex applications. Here, we report a novel multiplex RNA quantification method that uses capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) coupled with modified RT and asymmetric PCR. The reverse transcripts of seven in vitro transcribed RNAs were modified with common sequence tags and amplified by asymmetric PCR using primers specific to the common tags. The resulting amplicons were separated and quantified by CE-SSCP. A series of experiments using different amounts of RNA demonstrated that the assay had a limit of detection of 2 amol and a dynamic range of approximately 10(5). These results clearly indicate the potential of this method to provide robust and precise multiplex RNA quantification.

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Method validation of in vitro RNA transcript analysis on the Agilent 2100 Bioanalyzer

Cited by 23 publications

References 29 publications

Metabolic, pathologic, and genetic analysis of prostate tissues: quantitative evaluation of histopathologic and mRNA integrity after HR-MAS spectroscopy

Metabolic, pathologic, and genetic analysis of prostate tissues: quantitative evaluation of histopathologic and mRNA integrity after HR-MAS spectroscopy

Time-dependent expression of skeletal muscle troponin I mRNA in the contused skeletal muscle of rats: a possible marker for wound age estimation

Sensitive multiplex RNA quantification using capillary electrophoresis‐based single‐strand conformation polymorphism

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