21,508 research outputs found
A novel method for RNA extraction from FFPE samples reveals significant differences in biomarker expression between orthotopic and subcutaneous pancreatic cancer patient-derived xenografts.
Next-generation sequencing (NGS) can identify and validate new biomarkers of cancer onset, progression and therapy resistance. Substantial archives of formalin-fixed, paraffin-embedded (FFPE) cancer samples from patients represent a rich resource for linking molecular signatures to clinical data. However, performing NGS on FFPE samples is limited by poor RNA purification methods. To address this hurdle, we developed an improved methodology for extracting high-quality RNA from FFPE samples. By briefly integrating a newly-designed micro-homogenizing (mH) tool with commercially available FFPE RNA extraction protocols, RNA recovery is increased by approximately 3-fold while maintaining standard A260/A280 ratios and RNA quality index (RQI) values. Furthermore, we demonstrate that the mH-purified FFPE RNAs are longer and of higher integrity. Previous studies have suggested that pancreatic ductal adenocarcinoma (PDAC) gene expression signatures vary significantly under in vitro versus in vivo and in vivo subcutaneous versus orthotopic conditions. By using our improved mH-based method, we were able to preserve established expression patterns of KRas-dependency genes within these three unique microenvironments. Finally, expression analysis of novel biomarkers in KRas mutant PDAC samples revealed that PEAK1 decreases and MST1R increases by over 100-fold in orthotopic versus subcutaneous microenvironments. Interestingly, however, only PEAK1 levels remain elevated in orthotopically grown KRas wild-type PDAC cells. These results demonstrate the critical nature of the orthotopic tumor microenvironment when evaluating the clinical relevance of new biomarkers in cells or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to enhance and expand future FFPE-RNA-NGS cancer biomarker studies
Does strange kinetics imply unusual thermodynamics?
We introduce a fractional Fokker-Planck equation (FFPE) for Levy flights in
the presence of an external field. The equation is derived within the framework
of the subordination of random processes which leads to Levy flights. It is
shown that the coexistence of anomalous transport and a potential displays a
regular exponential relaxation towards the Boltzmann equilibrium distribution.
The properties of the Levy-flight FFPE derived here are compared with earlier
findings for subdiffusive FFPE. The latter is characterized by a
non-exponential Mittag-Leffler relaxation to the Boltzmann distribution. In
both cases, which describe strange kinetics, the Boltzmann equilibrium is
reached and modifications of the Boltzmann thermodynamics are not required
Use and Abuse of a Fractional Fokker-Planck Dynamics for Time-Dependent Driving
We investigate a subdiffusive, fractional Fokker-Planck dynamics occurring in
time-varying potential landscapes and thereby disclose the failure of the
fractional Fokker-Planck equation (FFPE) in its commonly used form when
generalized in an {\it ad hoc} manner to time-dependent forces. A modified FFPE
(MFFPE) is rigorously derived, being valid for a family of dichotomously
alternating force-fields. This MFFPE is numerically validated for a rectangular
time-dependent force with zero average bias. For this case subdiffusion is
shown to become enhanced as compared to the force free case. We question,
however, the existence of any physically valid FFPE for arbitrary varying
time-dependent fields that differ from this dichotomous varying family.Comment: 4 pages, 2 figure
Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples
Fragmented RNA from formalin-fixed paraffin-embedded (FFPE) tissue is a known obstacle to gene expression analysis. In this study, the impact of RNA integrity, gene-specific reverse transcription and targeted cDNA preamplification was quantified in terms of reverse transcription polymerase chain reaction (RT-qPCR) sensitivity by measuring 48 protein coding genes on eight duplicate cultured cancer cell pellet FFPE samples and twenty cancer tissue FFPE samples. More intact RNA modestly increased gene detection sensitivity by 1.6 fold (earlier detection by 0.7 PCR cycles, 95% CI = 0.593-0.850). Application of gene-specific priming instead of whole transcriptome priming during reverse transcription further improved RT-qPCR sensitivity by a considerable 4.0 fold increase (earlier detection by 2.0 PCR cycles, 95% CI = 1.73-2.32). Targeted cDNA preamplification resulted in the strongest increase of RT-qPCR sensitivity and enabled earlier detection by an average of 172.4 fold (7.43 PCR cycles, 95% CI = 6.83-7.05). We conclude that gene-specific reverse transcription and targeted cDNA preamplification are adequate methods for accurate and sensitive RT-qPCR based gene expression analysis of FFPE material. The presented methods do not involve expensive or complex procedures and can be easily implemented in any routine RT-qPCR practice
Quality of DNA extracted from formalin-fixed, paraffin-embedded canine tissues.
Veterinary pathology tissue banks are valuable resources for genetic studies. However, limited data exist as to whether quality DNA can be extracted from these tissues for use in canine genotyping studies. We extracted DNA from 44 formalin-fixed, paraffin-embedded (FFPE) tissue blocks from dogs; 9 of these dogs had DNA available from whole blood samples that had been banked. We genotyped DNA from 30 of 44 tissue blocks and 9 whole blood samples on the Illumina CanineHD BeadChip; DNA quality was insufficient in 14 of 44 samples from tissue blocks. There was significant correlation between the 260/280 ratio and single-nucleotide variation (SNV) call rate (p = 0.0276; r2 = 0.162); 23 of 30 samples from FFPE were genotyped with > 65% call rates. Median pairwise identical-by-state (IBS) analysis was 0.99 in 8 pairs of dogs with call rates > 65%. Neither age of tissue block nor specific tissue types were associated with significant differences in DNA concentration, 260/280 ratio, or SNV call rate. DNA extracted from tissue blocks can have variable quality, although comparable levels of homozygosity suggest that extracts from FFPE with call rates > 65% might provide similar results to samples from whole blood when analyzed on the Illumina CanineHD BeadChip
Linear Response in Complex Systems: CTRW and the Fractional Fokker-Planck Equations
We consider the linear response of systems modelled by continuous-time random
walks (CTRW) and by fractional Fokker-Planck equations under the influence of
time-dependent external fields. We calculate the corresponding response
functions explicitely. The CTRW curve exhibits aging, i.e. it is not
translationally invariant in the time-domain. This is different from what
happens under fractional Fokker-Planck conditions
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