Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: The SPIDIA experience.

In this note, we present an ad hoc procedure that combines qualitative (visual evaluation) and quantitative (ImageJ software) evaluations of Pulsed-Field Gel Electrophoresis (PFGE) images to assess the genomic DNA (gDNA) integrity of analyzed samples. This procedure could be suitable for the analysis of a large number of images by taking into consideration both the expertise of researchers and the objectiveness of the software. We applied this procedure on the first SPIDIA DNA External Quality Assessment (EQA) samples. Results show that the classification obtained by this ad hoc procedure allows a more accurate evaluation of gDNA integrity with respect to a single approach.

Data and performances evaluation of the SPIDIA-DNA Pan-European External Quality Assessment: 2nd SPIDIA-DNA laboratory report.

Within the EU-SPIDIA project (www.spidia.eu), the quality parameters of blood genomic DNA were defined [SPIDIA-DNA: an External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses - [1]; Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience - [2]; Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: the SPIDIA experience - [3]. DNA quality parameters were used to evaluate the laboratory performance within an External Quality Assessment (EQA) [Second SPIDIA-DNA External Quality Assessment (EQA): Influence of pre-analytical phase of blood samples on genomic DNA quality - [4]. These parameters included DNA purity and yield by UV spectrophotometric measurements, the presence of PCR interferences by Kineret software and genomic DNA integrity analysis by Pulsed Field Gel Electrophoresis. Here we present the specific laboratory report of the 2nd SPIDIA-DNA EQA as an example of data and performances evaluation.

Second SPIDIA-DNA External Quality Assessment (EQA): Influence of pre-analytical phase of blood samples on genomic DNA quality.

BACKGROUND: In order to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for DNA molecular testing, two pan-European External Quality Assessments (EQAs) were implemented within the European Commission funded project SPIDIA. Here we report the results of the 2nd SPIDIA EQA that has been implemented on the basis of the 1st DNA EQA with the inclusion of some stringent conditions related to blood storage temperature and time. METHODS: SPIDIA facility sent to all the participants the same blood sample to be processed by their own procedure following SPIDIA suggestion for time and temperature storage. Evaluated genomic DNA (gDNA) quality parameters were: purity and yield by UV spectrophotometric analysis, PCR interferences by Kineret software and integrity by a dedicated algorithm. RESULTS/CONCLUSIONS: 188 applications have been collected from 26 European countries. A high variability of gDNA integrity was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A dedicated analysis on pre-analytical variables and the evaluated gDNA quality parameters showed that blood storage and DNA extraction procedures influence gDNA integrity. The performances of the participants were improved in comparison with the 1st SPIDIA-DNA EQA, probably due to adopted more stringent pre-analytical conditions.

PAPC mediates self/non-self-distinction during Snail1-dependent tissue separation.

Cleft-like boundaries represent a type of cell sorting boundary characterized by the presence of a physical gap between tissues. We studied the cleft-like ectoderm-mesoderm boundary in Xenopus laevis and zebrafish gastrulae. We identified the transcription factor Snail1 as being essential for tissue separation, showed that its expression in the mesoderm depends on noncanonical Wnt signaling, and demonstrated that it enables paraxial protocadherin (PAPC) to promote tissue separation through two novel functions. First, PAPC attenuates planar cell polarity signaling at the ectoderm-mesoderm boundary to lower cell adhesion and facilitate cleft formation. Second, PAPC controls formation of a distinct type of adhesive contact between mesoderm and ectoderm cells that shows properties of a cleft-like boundary at the single-cell level. It consists of short stretches of adherens junction-like contacts inserted between intermediate-sized contacts and large intercellular gaps. These roles of PAPC constitute a self/non-self-recognition mechanism that determines the site of boundary formation at the interface between PAPC-expressing and -nonexpressing cells.