Effect of RNA quality on transcript intensity levels in microarray analysis of human post-mortem brain tissues

Popova, Tatiana; Mennerich, Detlev; Weith, Andreas; Quast, Karsten

doi:10.1186/1471-2164-9-91

Cited by 68 publications

(63 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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%

Improved RNA preservation for immunolabeling and laser microdissection

Brown

Dw²

2009

RNA

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Improved RNA preservation for immunolabeling and laser microdissection

Brown

Dw²

2009

RNA

View full text Add to dashboard Cite

show abstract

“…Identifying the genes still expressed just prior to cell death meant working with RNA samples exhibiting a certain degree of degradation. Several recent studies have looked at how much RNA degradation could influence gene expression measurements, but their conclusions remain a matter of debate (36)(37)(38). According to Opitz et al (36), samples with low RNA quality were still valuable for gene expression analysis but their use generated a greater variability between samples and an underestimation of genes with low transcript levels.…”

Section: Discussionmentioning

confidence: 99%

Whole-Genome Transcriptional Analysis of Escherichia coli during Heat Inactivation Processes Related to Industrial Cooking

Guernec

Robichaud-Rincon

Saucier

2013

Appl Environ Microbiol

View full text Add to dashboard Cite

Escherichia coli K-12 was grown to the stationary phase, for maximum physiological resistance, in brain heart infusion (BHI) broth at 37°C. Cells were then heated at 58°C or 60°C to reach a process lethality value (F o 70 10 ) of 2 or 3 or to a core temperature of 71°C (control industrial cooking temperature). Growth recovery and cell membrane integrity were evaluated immediately after heating, and a global transcription analysis was performed using gene expression microarrays. Only cells heated at 58°C with F o ‫؍‬ 2 were still able to grow on liquid or solid BHI broth after heat treatment. However, their transcriptome did not differ from that of bacteria heated at 58°C with F o ‫؍‬ 3 (P value for the false discovery rate [P-FDR] > 0.01), where no growth recovery was observed posttreatment. Genome-wide transcriptomic data obtained at 71°C were distinct from those of the other treatments without growth recovery. Quantification of heat shock gene expression by real-time PCR revealed that dnaK and groEL mRNA levels decreased significantly above 60°C to reach levels similar to those of control cells at 37°C (P < 0.0001). Furthermore, despite similar levels of cell inactivation measured by growth on BHI media after heating, 132 and 8 genes were differentially expressed at 71°C compared to 58°C and 60°C at F o ‫؍‬ 3, respectively (P-FDR < 0.01). Among them, genes such as aroA, citE, glyS, oppB, and asd, whose expression was upregulated at 71°C, may be worth investigating as good biomarkers for accurately determining the efficiency of heat treatments, especially when cells are too injured to be enumerated using growth media.

show abstract

“…A subgroup of mRNA transcripts carrying the 3′-UTR AUUUA motif was shown to be particularly susceptible to PMIrelated degradation in animal models (Catts et al, 2005). In addition, a systemic analysis of postmortem brain tissues also demonstrated that RNA quality is a more critical determinant in analyzing a broad spectrum of mRNA transcripts, even more so than agony factors (Popova et al, 2008). Thus, data normalization to endogenous housekeeping reference genes without regard to RNA quality effects could be misleading.…”

Section: Discussionmentioning

confidence: 99%

“…To test this idea, we primarily focus on brain tissues because neuronal cells abundantly express HSPs and are highly vulnerable to a variety of cellular stresses, such as hypoxia/anoxia as well as disturbances in energy homeostasis (Hecker and McGarvey, 2011;Stetler et al, 2010). Furthermore, brain tissues also have a practical advantage in postmortem RNA profiling; it has been demonstrated that postmortem gene transcripts in brain tissues are relatively stable and intact with longer PMI, and are thus suitable for extraction and quantitative analyses of RNA (Heinrich et al, 2007a;2007b;Leonard et al, 1993;Popova et al, 2008). Therefore, the present study aims to quantitatively analyze a subgroup of brain-enriched HSP mRNA transcripts particularly in the postmortem human occipital lobes, thus gaining useful clues on their differential profiles by the cause of death, such as traumatic injury (TI), mechanical asphyxiation (ASP), or sudden cardiac death (SCD).…”

Section: Introductionmentioning

confidence: 99%

Quantitative Analyses of Postmortem Heat Shock Protein mRNA Profiles in the Occipital Lobes of Human Cerebral Cortices: Implications in Cause of Death

Chung

Seo

Kim

et al. 2012

Molecules and Cells

View full text Add to dashboard Cite

Quantitative RNA analyses of autopsy materials to diagnose the cause and mechanism of death are challenging tasks in the field of forensic molecular pathology. Alterations in mRNA profiles can be induced by cellular stress responses during supravital reactions as well as by lethal insults at the time of death. Here, we demonstrate that several gene transcripts encoding heat shock proteins (HSPs), a gene family primarily responsible for cellular stress responses, can be differentially expressed in the occipital region of postmortem human cerebral cortices with regard to the cause of death. HSPA2 mRNA levels were higher in subjects who died due to mechanical asphyxiation (ASP), compared with those who died by traumatic injury (TI). By contrast, HSPA7 and A13 gene transcripts were much higher in the TI group than in the ASP and sudden cardiac death (SCD) groups. More importantly, relative abundances between such HSP mRNA species exhibit a stronger correlation to, and thus provide more discriminative information on, the death process than does routine normalization to a housekeeping gene. Therefore, the present study proposes alterations in HSP mRNA composition in the occipital lobe as potential forensic biological markers, which may implicate the cause and process of death.

show abstract

Effect of RNA quality on transcript intensity levels in microarray analysis of human post-mortem brain tissues

Cited by 68 publications

References 31 publications

Improved RNA preservation for immunolabeling and laser microdissection

Improved RNA preservation for immunolabeling and laser microdissection

Whole-Genome Transcriptional Analysis of Escherichia coli during Heat Inactivation Processes Related to Industrial Cooking

Quantitative Analyses of Postmortem Heat Shock Protein mRNA Profiles in the Occipital Lobes of Human Cerebral Cortices: Implications in Cause of Death

Contact Info

Product

Resources

About