A laser microdissection-based workflow for FFPE tissue microproteomics: Important considerations for small sample processing

Methods ◽  
2016 ◽  
Vol 104 ◽  
pp. 154-162 ◽  
Author(s):  
Rémi Longuespée ◽  
Deborah Alberts ◽  
Charles Pottier ◽  
Nicolas Smargiasso ◽  
Gabriel Mazzucchelli ◽  
...  
Keyword(s):  
Laser Microdissection ◽  
Small Sample ◽  
Ffpe Tissue ◽  
Sample Processing

scholarly journals Validation of MALDI-MS imaging data of selected membrane lipids in murine brain with and without laser postionization by quantitative nano-HPLC-MS using laser microdissection

2020 ◽  
Vol 412 (25) ◽  
pp. 6875-6886 ◽  
Author(s):  
Fabian B. Eiersbrock ◽  
Julian M. Orthen ◽  
Jens Soltwisch
Keyword(s):  
Signal Intensity ◽  
Laser Microdissection ◽  
Membrane Lipids ◽  
Chemical Properties ◽  
Small Sample ◽  
Tissue Type ◽  
Imaging Data ◽  
Response Factors ◽  
Lipid Species ◽  
Intensity Response

Abstract MALDI mass spectrometry imaging (MALDI-MSI) is a widely used technique to map the spatial distribution of molecules in sectioned tissue. The technique is based on the systematic generation and analysis of ions from small sample volumes, each representing a single pixel of the investigated sample surface. Subsequently, mass spectrometric images for any recorded ion species can be generated by displaying the signal intensity at the coordinate of origin for each of these pixels. Although easily equalized, these recorded signal intensities, however, are not necessarily a good measure for the underlying amount of analyte and care has to be taken in the interpretation of MALDI-MSI data. Physical and chemical properties that define the analyte molecules’ adjacencies in the tissue largely influence the local extraction and ionization efficiencies, possibly leading to strong variations in signal intensity response. Here, we inspect the validity of signal intensity distributions recorded from murine cerebellum as a measure for the underlying molar distributions. Based on segmentation derived from MALDI-MSI measurements, laser microdissection (LMD) was used to cut out regions of interest with a homogenous signal intensity. The molar concentration of six exemplary selected membrane lipids from different lipid classes in these tissue regions was determined using quantitative nano-HPLC-ESI-MS. Comparison of molar concentrations and signal intensity revealed strong deviations between underlying concentration and the distribution suggested by MSI data. Determined signal intensity response factors strongly depend on tissue type and lipid species.

Quantitative nuclease protection assay (qNPA) for gene expression analysis on breast cancer core biopsies.

2011 ◽  
Vol 29 (27_suppl) ◽  
pp. 49-49 ◽  
Author(s):  
M. C. Evangelist ◽  
J. Snider ◽  
J. Krushkal ◽  
Y. Qu ◽  
A. Kulkarni ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Small Sample Size ◽  
Small Sample ◽  
Ffpe Tissue ◽  
Cancer Tissue ◽  
Breast Cancer Patients ◽  
Fresh Tissue ◽  
Interacting Protein ◽  
Oncogenic Pathways

49 Background: qNPA employs in situ hybridization of detection probes to cross linked mRNA, making it ideal for formalin fixed paraffin embedded (FFPE) tissue. It has been shown to measure gene expression in archived lymphoid and lung cancer tissue. We assessed the feasibility of qNPA to measure differentially expressed genes in pretreatment FFPE core breast biopsies among pathologic responders (pR) and nonresponders (pNR) to preoperative chemotherapy. Methods: We included preoperative breast cancer patients treated at our institution from 2003-09 with FFPE core biopsies. mRNA expression of 170 genes, representing oncogenic pathways or associated with anthracycline and taxane response was measured by qNPA (HTG, Tucson, AZ). Data was normalized to 3 housekeeper genes and average of 3 biologic replicates reported. Seven genes below detection in > 50% samples were excluded. Expression values of 163 unique genes were analyzed for pR vs pNR with dChip software. Empirical FDR was estimated using 1,000 permutations of sample labels. Results: Treatment and response: Sample failure: 6/57 (10%). pR vs pNR did not separate on hierarchical clustering. FLJ12650 and IGFBP2 showed lower expression in pR vs pNR with fold changes of 4.09 and 2.40, respectively (p < 0.01; median FDR: 1/163). FLJ12650 was significant (p < 0.01, median FDR: 0/163) when patients receiving anthracycline ± taxane were analyzed (groups 1-3) and showed a trend (p < 0.05) in group 1 alone. Conclusions: qNPA for limited available FFPE tissue from core biopsies is feasible with acceptable sample failure rates. Small sample size and number of analyzed genes limited definitive conclusion about informative genes in our study. Our FLJ12650 results, a gene coding for membrane Na+/K+ ATPase interacting protein, are consistent with previous findings of overexpression in pNR to anthracyclines + taxanes (Hess et al, JCO 2006, Vol 24; 4236) using fresh tissue. Future qNPA validation of predictive markers, identified by whole transcriptome analysis in a homogenous cohort may provide more definitive results. [Table: see text]

scholarly journals Blank Assessment for Ultra-Small Radiocarbon Samples: Chemical Extraction and Separation Versus AMS

Radiocarbon ◽  
2010 ◽  
Vol 52 (3) ◽  
pp. 1322-1335 ◽  
Author(s):  
Guaciara M Santos ◽  
John R Southon ◽  
Nicholas J Drenzek ◽  
Lori A Ziolkowski ◽  
Ellen Druffel ◽  
...  
Keyword(s):  
Small Sample ◽  
Small Samples ◽  
Chemical Extraction ◽  
Mass Ratio ◽  
Sample Analysis ◽  
Sample Processing ◽  
Extraction And Separation ◽  
Research Activities ◽  
Processing Techniques ◽  
The University

The Keck Carbon Cycle AMS facility at the University of California, Irvine (KCCAMS/UCI) has developed protocols for analyzing radiocarbon in samples as small as ∼0.001 mg of carbon (C). Mass-balance background corrections for modern and 14C-dead carbon contamination (MC and DC, respectively) can be assessed by measuring 14C-free and modern standards, respectively, using the same sample processing techniques that are applied to unknown samples. This approach can be validated by measuring secondary standards of similar size and 14C composition to the unknown samples. Ordinary sample processing (such as ABA or leaching pretreatment, combustion/graphitization, and handling) introduces MC contamination of ∼0.6 ± 0.3 μg C, while DC is ∼0.3 ± 0.15 μg C. Today, the laboratory routinely analyzes graphite samples as small as 0.015 mg C for external submissions and ≅0.001 mg C for internal research activities with a precision of ∼1% for ∼0.010 mg C. However, when analyzing ultra-small samples isolated by a series of complex chemical and chromatographic methods (such as individual compounds), integrated procedural blanks may be far larger and more variable than those associated with combustion/graphitization alone. In some instances, the mass ratio of these blanks to the compounds of interest may be so high that the reported 14C results are meaningless. Thus, the abundance and variability of both MC and DC contamination encountered during ultra-small sample analysis must be carefully and thoroughly evaluated. Four case studies are presented to illustrate how extraction chemistry blanks are determined.

scholarly journals Ultra-Microscale (5–25 μg C) Analysis of Individual Lipids by 14C AMS: Assessment and Correction for Sample Processing Blanks

Radiocarbon ◽  
2007 ◽  
Vol 49 (1) ◽  
pp. 69-82 ◽  
Author(s):  
Sunita R Shah ◽  
Ann Pearson
Keyword(s):  
Water Column ◽  
Euphotic Zone ◽  
Total Sample ◽  
Small Sample ◽  
Sample Processing ◽  
Central Pacific ◽  
The North ◽  
Pure Compounds ◽  
Small Sample Sizes ◽  
Different Sources

Measurements of the natural abundance of radiocarbon in biomarker molecules can be used to elucidate the biogeochemical roles of marine bacteria and archaea in the oceanic water column. However, the relatively low concentration of biomass, especially below the euphotic zone, inevitably results in small sample sizes for compound-specific analyses. In ultra-microscale Δ14C measurements, which we define as measurements on samples smaller than 25 μg C, the process of isolating pure compounds and preparing them for measurement adds significant background carbon. This additional blank carbon can contribute up to 40% of the total sample mass; therefore, it is necessary to quantify all components of the processing blank in order to make appropriate corrections. Complete propagation of error is critical in order to report the correct analytical uncertainty. The carbon blank is composed of at least 3 different sources: i) those that scale in proportion to the mass of the sample; ii) sources that contribute a constant mass of blank, e.g. closed-tube combustion; and iii) contaminants from vacuum lines and/or other aspects of sample handling that are difficult to quantify. We approached the problem of correcting for the total sample processing blank by deriving a 4-part isotopic mass balance based on separating the 3 exogenous components from the sample. Subsequently, we derived the appropriate equations for the full propagation of error associated with these corrections. Equations for these terms are presented. Full treatment of a set of raw data is demonstrated using compound-specific Δ14C data from the North Central Pacific water column.

scholarly journals Progress in AMS Target Production of Sub-Milligram Samples at the NERC Radiocarbon Laboratory

Radiocarbon ◽  
2005 ◽  
Vol 47 (3) ◽  
pp. 453-464 ◽  
Author(s):  
Tanya Ertunç ◽  
Sheng Xu ◽  
Charlotte L Bryant ◽  
Colin Maden ◽  
Callum Murray ◽  
...  
Keyword(s):  
Environmental Samples ◽  
High Vacuum ◽  
Small Sample ◽  
Environmental Research ◽  
Research Centre ◽  
Sample Processing ◽  
Graphite Target ◽  
Vacuum Line ◽  
Technical Details ◽  
Target Production

Recent progress in graphite target production for sub-milligram environmental samples in our facility is presented. We describe an optimized hydrolysis procedure now routinely used for the preparation of CO2 from inorganic samples, a new high-vacuum line dedicated to small sample processing (combining sample distillation and graphitization units), as well as a modified graphitization procedure. Although measurements of graphite targets as small as 35 μg C have been achieved, system background and measurement uncertainties increase significantly below 150 μg C. As target lifetime can become critically short for targets <150 μg C, the facility currently only processes inorganic samples down to 150 μg C. All radiocarbon measurements are made at the Scottish Universities Environmental Research Centre (SUERC) accelerator mass spectrometry (AMS) facility. Sample processing and analysis are labor-intensive, taking approximately 3 times longer than samples ≥ 500 μg C. The technical details of the new system, graphitization yield, fractionation introduced during the process, and the system blank are discussed in detail.

scholarly journals Micro-Extraction of Chlordiazepoxide and its Primary Metabolites, Desmethylchlordiazepoxide and Demoxepam, from Plasma and Their Measurement by Liquid Chromatography

1998 ◽  
Vol 35 (4) ◽  
pp. 528-533 ◽  
Author(s):  
Deborah J Garretty ◽  
Kim Wolff ◽  
Alastair W M Hay
Keyword(s):  
Liquid Chromatography ◽  
Limit Of Detection ◽  
Plasma Concentrations ◽  
Extraction Procedure ◽  
Sample Volume ◽  
Small Sample ◽  
Sample Processing ◽  
Primary Metabolites ◽  
Sample Handling ◽  
Coefficients Of Variation

We have developed a micro-extraction procedure for the analysis of chlordiazepoxide and its two unique metabolites, demoxepam and desmethylchlordiazepoxide, in plasma, using liquid chromatography. The method is both reliable and sensitive for the quantitation of low plasma concentrations of these three compounds. The extraction procedure allows rapid sample processing, which, together with the small sample volume (100 μL), makes it ideal for routine sample handling. The limit of detection for the three compounds ranged from 0.075 to 0.125 mg/L and recovery of the three different benzodiazepines ranged from 87 to 94%. Within- and between-assay coefficients of variation ranged from 3.1–4.5% and from 4.7 to 7.6%, respectively.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

Validating the Factor Structure of the Stigma of Suicide Scale–Short Form Spanish Version Among Healthcare Students

Crisis ◽  
2020 ◽  
pp. 1-5
Author(s):  
Ruthmarie Hernández-Torres ◽  
Paola Carminelli-Corretjer ◽  
Nelmit Tollinchi-Natali ◽  
Ernesto Rosario-Hernández ◽  
Yovanska Duarté-Vélez ◽  
...  
Keyword(s):  
Factor Structure ◽  
Structural Equation ◽  
Short Form ◽  
Small Sample Size ◽  
Small Sample ◽  
Spanish Version ◽  
Equation Modeling ◽  
Cross Sectional ◽  
Healthcare Students ◽  
Spanish Speaking

Abstract. Background: Suicide is a leading cause of death among Spanish-speaking individuals. Suicide stigma can be a risk factor for suicide. A widely used measure is the Stigma of Suicide Scale-Short Form (SOSS-SF; Batterham, Calear, & Christensen, 2013 ). Although the SOSS-SF has established psychometric properties and factor structure in other languages and cultural contexts, no evidence is available from Spanish-speaking populations. Aim: This study aims to validate a Spanish translation of the SOSS-SF among a sample of Spanish-speaking healthcare students ( N = 277). Method: We implemented a cross-sectional design with quantitative techniques. Results: Following a structural equation modeling approach, a confirmatory factor analysis (CFA) supported the three-factor model proposed by Batterham and colleagues (2013) . Limitations: The study was limited by the small sample size and recruitment by availability. Conclusion: Findings suggest that the Spanish version of the SOSS-SF is a valid and reliable tool with which to examine suicide stigma among Spanish-speaking populations.

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