Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA

Thompson, Karol L.; Pine, P. Scott; Rosenzweig, Barry A.; Turpaz, Yaron; Retief, Jacques D.

doi:10.1186/1472-6750-7-57

Cited by 78 publications

(83 citation statements)

References 21 publications

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“…For example, after 5 min in PBS there was a gene-dependent five-to 22-fold reduction in transcript level relative to fresh-frozen control ( Fig. 2 Thompson et al 2007). Surprisingly, 30-min PBS incubation did not result in a proportional reduction in transcripts relative to that found for 5-min PBS.…”

Section: Discussionmentioning

confidence: 99%

“…Genomics approaches continue to be criticized because of inconsistent results (Miklos and Maleszka 2004;Draghici et al 2006), and while some of this inconsistency arises from data processing (Shi et al 2008), differences in sample RNA integrity markedly affect transcript quantification and likely are the cause of much of the inconsistency (Auer et al 2003;Lipska et al 2006;Atz et al 2007;Thompson et al 2007;Popova et al 2008). Sample RNA of the highest quality is therefore imperative.…”

Section: Discussionmentioning

confidence: 99%

“…It is now clearly evident that if meaningful transcriptome analyses, whether at the single transcript or whole transcriptome level, are to be obtained, use of high quality RNA is critical (Auer et al 2003;Imbeaud et al 2005;Fleige and Pfaffl 2006;Lipska et al 2006;Nolan et al 2006;Copois et al 2007;Thompson et al 2007). Genomics approaches continue to be criticized because of inconsistent results (Miklos and Maleszka 2004;Draghici et al 2006), and while some of this inconsistency arises from data processing (Shi et al 2008), differences in sample RNA integrity markedly affect transcript quantification and likely are the cause of much of the inconsistency (Auer et al 2003;Lipska et al 2006;Atz et al 2007;Thompson et al 2007;Popova et al 2008).…”

Section: Discussionmentioning

confidence: 99%

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Improved RNA preservation for immunolabeling and laser microdissection

Brown

Dw²

2009

RNA

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Microdissection techniques have the potential to allow for transcriptome analyses in specific populations of cells that are isolated from heterogeneous tissues such as the nervous system and certain cancers. Problematically, RNA is not stable under the labeling conditions usually needed to identify the cells of interest for microdissection. We have developed an immunolabeling method that utilizes a high salt buffer to stabilize RNA during prolonged antibody incubations. We first assessed RNA integrity by three methods and found that tissue incubated in high salt buffer for at least 20 h yielded RNA of similar quality to that for RNA extracted from fresh-frozen tissue, which is considered highest quality. Notably, the integrity was superior to that for RNA extracted from tissue processed using rapid immunolabeling procedures (5 min total duration). We next established that high salt buffer was compatible with immunolabeling, as demonstrated by immunofluorescent detection of dopamine neurons in the brain. Finally, we laser microdissected dopamine neurons that were immunolabeled using high salt buffer and demonstrated that RNA integrity was preserved. Our described method yields high quality RNA from immunolabeled microdissected cells, an essential requirement for meaningful genomics investigations of normal and pathological cells isolated from complex tissues.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Improved RNA preservation for immunolabeling and laser microdissection

Brown

Dw²

2009

RNA

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“…However, in an article about quality control of RNA for microarray, RIN of 7.0 was used as a cut-off value for deciding whether the RNA is suitable for microarray analysis [31]. The authors selected this value because Thompson et al showed that there was a substantial increase in the rate of false positives on the microarray when the starting RNA had a RIN value of <7.0 [32]. This may suggest the RNA obtained in our study which has RIN value of 7.5 may also be suitable for microarray analysis.…”

Section: Yields Of Rna From Rat Np Tissuementioning

confidence: 99%

Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation

2015

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Reverse-transcription quantitative PCR (RT-qPCR) is a powerful technique for quantification of gene expression. Extraction of RNA directly from human and large animal intervertebral disc (IVD) tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. IVD is consisted of nucleus pulposus (NP) and annulus fibrosis (AF). We compared the yield and quality of RNA extracted from cultured cells using TRIzol and combined TRIzol-column method (TRIspin) with and without bovine NP tissue added for high proteoglycan content simulation. Higher yields were obtained using the TRIzol method compared to the TRIspin method and the quality of RNA extracted using both methods was comparable. Cryosectioning was by far the most effective homogenization method for the tough bovine NP tissue. The typical modification to TRIzol extraction by the use of high salt solution in a previously reported study for tissue with high proteoglycan was insufficient for bovine NP tissue, where undesirable precipitates were formed during the RNA precipitation step. This necessitates other modifications to the protocol. In our study, the combination of additional phase separation and high salt precipitation was shown to be effective to avoid the formation of the undesirable precipitates. The modified TRIzol method was compared with the TRIspin method and shown to give higher RNA yield which resulted in smaller Ct values in RT-qPCR. In addition, we showed that the cryosectioning method was applicable to bovine muscle, mouse IVD and rat NP tissues. This method of RNA preparation also allows simultaneous preparation of tissue for histological study and quantitative gene expression analysis.

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“…However, in an article about quality control of RNA for microarray, RIN of 7.0 was used as a cut-off value for deciding whether the RNA is suitable for microarray analysis [149]. The authors selected this value because Thompson et al showed that there was a substantial increase in the rate of false positives on the microarray when the starting RNA had a RIN value of < 7.0 [150]. This may suggest the RNA obtained in our study which has RIN value of 7.5 may also be suitable for microarray analysis.…”

Section: Yield Of Rna From Rat Np Tissuementioning

confidence: 99%

Technique and method development for intervertebral disc (IVD) research

Lee¹,

李芷恩²

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Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. The direct and indirect cost of managing back pain posts heavy socioeconomic burden to the society. Improved technologies and techniques together with well documented experiments will benefit the research field by saving effort in doing the optimization in every laboratory and avoiding experiment failure due to incomplete understanding of the procedures. These can accelerate scientific discovery, reduce the sacrifice of animals and enable a more effective use of funding.Nucleus pulposus (NP) is the central part of IVD. Differences in matrix compositions in human NP clinical samples demand different cell isolation protocols for optimal results but there is no clear guide about this to date. Sub-optimal protocols may result in low cell yield, limited reliability of results or even failure of experiments. We experimented different isolation protocols to study different parameters involved and suggested some rules for cell isolation in three main applications: RNA extraction for phenotyping, cell isolation for cell culture, and characterization by flow cytometry.In addition, instead of extracting RNA from isolated cells, extraction of RNA from tissues directly may avoid the change of RNA levels during the cell isolation process.However, extraction of RNA directly from human and large animal IVD tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. Thus we developed a method for RNA extraction from bovine disc tissues by integrating the use of cryosectioning, additional phase separation and high salt precipitation into conventional guanidinium thiocyanate based method. With this method, RNA could be extracted from the NP tissue directly but the concentration was low. A shift toward 270 nm was observed in its UV spectrum which was due to phenol contamination. This caused an overestimation of RNA concentration. Hence we developed a computational method based on UV spectra for correcting the In short, different methods related to IVD research were developed and optimized. With improved methods together with a better understanding of the underlying rationale, researchers can save time and cost in their experiments and reduce experiment failure rate. This will help to accelerate researches. New methods also enable studies which were not feasible in the past.(484 words)

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Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA

Cited by 78 publications

References 21 publications

Improved RNA preservation for immunolabeling and laser microdissection

Improved RNA preservation for immunolabeling and laser microdissection

Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation

Technique and method development for intervertebral disc (IVD) research

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