Novel zinc-based fixative for high quality DNA, RNA and protein analysis

We have developed a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity. The effects of 25 different fixative recipes on the fixed quality of tissues from C57BL/6 mice were investigated. Results from IHC, PCR, RT–PCR, RNA Agilent Bioanalyser and Real-Time PCR showed that a novel zinc-based fixative (Z7) containing zinc trifluoroacetate, zinc chloride and calcium acetate was significantly better than the standard zinc-based fixative (Z2) and neutral buffered formalin (NBF) for DNA, RNA and protein preservation. DNA sequences up to 2.4 kb in length and RNA fragments up to 361 bp in length were successfully amplified from Z7 fixed tissues, as demonstrated by PCR, RT–PCR and Real-Time PCR. Total protein analysis was achieved using 2-D gel electrophoresis. In addition, nucleic acids and proteins were very stable over a 6–14-month period. This improved, non-toxic and economical tissue fixative could be applied for routine use in pathology laboratories to permit subsequent genomic/proteomic studies

A Rapid and Highly Sensitive Method of Non Radioactive Colorimetric In Situ Hybridization for the Detection of mRNA on Tissue Sections

Background: Non Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the analysis of genes expressed at low levels. Methodology/Principal Findings: A stable, non-toxic, zinc-based fixative was tested in NoRISH experiments on sections of mouse embryos using four probes (Lhx6, Lhx7, ncapg and ret) that have different spatial patterns and expression levels. We showed that Z7 can successfully replace paraformaldehyde used so far for tissue fixation in NoRISH; the morphology of the cryosections of Z7-fixed tissues was excellent, and the fixation time required for tissues sized 1 cm was 1 hr instead of 24 hr for paraformaldehyde. The hybridization signal on the sections of the Z7-treated embryos always appeared earlier than that of the PFA-fixed embryos. In addition, a 50–60 % shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method. Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFAfixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method. Conclusions/Significance: Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highl

Tracing Cattle Breeds with Principal Components Analysis Ancestry Informative SNPs

The recent release of the Bovine HapMap dataset represents the most detailed survey of bovine genetic diversity to date, providing an important resource for the design and development of livestock production. We studied this dataset, comprising more than 30,000 Single Nucleotide Polymorphisms (SNPs) for 19 breeds (13 taurine, three zebu, and three hybrid breeds), seeking to identify small panels of genetic markers that can be used to trace the breed of unknown cattle samples. Taking advantage of the power of Principal Components Analysis and algorithms that we have recently described for the selection of Ancestry Informative Markers from genomewide datasets, we present a decision-tree which can be used to accurately infer the origin of individual cattle. In doing so, we present a thorough examination of population genetic structure in modern bovine breeds. Performing extensive cross-validation experiments, we demonstrate that 250-500 carefully selected SNPs suffice in order to achieve close to 100% prediction accuracy of individual ancestry, when this particular set of 19 breeds is considered. Our methods, coupled with the dense genotypic data that is becoming increasingly available, have the potential to become a valuable tool and have considerable impact in worldwide livestock production. They can be used to inform the design of studies of the genetic basis of economically important traits in cattle, as well as breeding programs and efforts to conserve biodiversity. Furthermore, the SNPs that we have identified can provide a reliable solution for the traceability of breed-specific branded products

Development of a zinc-based fixative for DNA, RNA and protein molecular studies

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Η δημιουργία ενός καινοτόμου μονιμοποιητικού ιστών και κυττάρων με βάση τον ψευδάργυρο, που επιτρέπει την υψηλού επιπέδου μοριακή ανάλυση DNA, RNA και πρωτεϊνών

Fixation is a chemical process by which tissue and cell components are ‘stabilised’ temporally and spatially, preserving morphology, nucleic acids, proteins and other cell constituents. There is a wide range of fixatives, based on their chemical function, but none of the compounds used is ideal, i.e. for preserving both morphology and integrity of DNA, RNA and protein. This thesis aimed to develop a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity. The effects of 30 different fixative recipes on the fixed quality of tissues from C57B16 mice colon, liver and spleen were investigated. Results from immunohistochemistry (IHC), polymerase chain reaction (PCR), Reverse transcription PCR, RNA Agilent Bioanalyser and Real-Time PCR showed that a novel zinc-based fixative (Z7) containing zinc trifluoroacetate, zinc chloride and calcium acetate was significantly better than the standard zinc based fixative (Z2) and neutral buffered formalin (NBF) for DNA, RNA and protein preservation. DNA sequences up to 2.4Kb in length and RNA fragments up to 361 bp in length were successfully amplified from Z7 fixed tissues, as demonstrated by PCR, RT-PCR and Real-Time PCR. Total protein analysis was achieved using 2-D gel electrophoresis. In addition, nucleic acids and proteins were very stable in fixed tissue blocks over a 12 month period. Fresh frozen samples were also included in this experimental model as controls for DNA and RNA integrity. Using Affymetrix exon microarrays it was shown that Z7-fixed samples had no statistical significant difference in the exon alternative splicing pattern compared to fresh-frozen samples, and could be used for further human cancer array genome studies. Mass Spectroscopy studies using synthetic peptides and their reaction with zinc trifluoroacetate showed that zinc is binding to at least one amino acid, either cysteine or histidine. Future work is required to elucidate the exact chemical mechanism of zinc fixation.In conclusion, this novel, non-toxic and economical tissue fixative could be applied for routine laboratory use to permit subsequent genomic/proteomic studies.Η μονιμοποίηση είναι μια πολύπλοκη βιοχημική διαδικασία με την οποία τμήματα ιστού και κυττάρων σταθεροποιούνται στο χώρο, συντηρώντας τη μορφολογία, τα νουκλεϊνικά οξέα, τις πρωτεΐνες και άλλα συστατικά κυττάρων. Υπάρχει ένα ευρύ φάσμα μονιμοποιητικών, ανάλογα με την βιοχημική τους λειτουργία, αλλά κανένα από αυτά δεν είναι “ιδανικό”, δηλαδή ικανό να προσφέρει διατήρηση της μορφολογίας των ιστών η των κυττάρων, της ακεραιότητας του DNA, του RNA καθώς και των πρωτεϊνών των ιστών η των κυττάρων. Αυτή η διατριβή στόχευσε να αναπτύξει ένα αξιόπιστο, οικονομικώς αποδοτικό και μη τοξικό μονιμοποιητικό για να ικανοποιήσει τις ανάγκες της σύγχρονης μοριακής παθοβιολογικής έρευνας, ιδιαίτερα για την ακεραιότητα του DNA και RNA. Τα αποτελέσματα 35 διαφορετικών μονιμοποιητικών στη ποιότητα των ιστών από 5 διαφορετικά όργανα ποντικιών C57Bl6 ερευνήθηκαν. Τα αποτελέσματα πειραμάτων με την χρήση των τεχνικών ανοσοϊστοχημείας, αλυσιδωτής αντίδρασης πολυμεράσης (PCR), RT-PCR, RNA Agilent Bioanalyser και Real-time PCR έδειξαν ότι ένα νέο μονιμοποιητικό με βάση τον ψευδάργυρο (Z7) που περιέχει τριφθοροοξικό ψευδάργυρο, χλωριούχο ψευδάργυρο και οξικό άλας ασβεστίου ήταν σημαντικά καλύτερο από το τυποποιημένο βασισμένο στον ψευδάργυρο μονιμοποιητικό (Z2) και την neutral buffered formalin (NBF) για το DNA, το RNA και την πρωτεϊνική διατήρηση. Ακολουθίες DNA μήκους μέχρι 2.4 Kb και RNA μήκους 361 bp ενισχύθηκαν επιτυχώς από Z7 μονιμοποιημένους ιστούς, σύμφωνα με τα αποτελέσματα των πειραμάτων με τις τεχνικές PCR, RT-PCR και Real-time PCR. Η ανάλυση ολικής πρωτεΐνης από δείγματα ιστών επιτεύχθηκε χρησιμοποιώντας τη 2D-PAGE ηλεκτροφόρηση. Επιπλέον, τα νουκλεϊνικά οξέα και οι πρωτεΐνες διατηρήθηκαν εξίσου καλά σε Ζ7 μονιμοποιημένους ιστούς ακόμη και μετά την πάροδο χρονικής περιόδου 18 μηνών. “Φρέσκα- παγωμένα” δείγματα περιλήφθηκαν επίσης σε αυτό το πειραματικό πρότυπο ως μάρτυρες (controls) για την ακεραιότητα DNA, RNA και πρωτεϊνών. Η χρησιμοποίηση της τεχνικής Affymetrix exon microarrays έδειξε ότι τα Ζ7 μονιμοποιημένα δείγματα από ιστούς ποντικιών, δεν είχαν καμία στατιστική σημαντική διαφορά σε όλο το γονιδίωμα όσων αφορά την έκφραση των γονιδίων όσο και το εναλλακτικό μάτισμα των εξωνίων έναντι των “φρέσκων-παγωμένων” δειγμάτων. Συνεπώς, το Ζ7 μονιμοποιητικό θα μπορούσε να χρησιμοποιηθεί για περαιτέρω μελέτες χρησιμοποιώντας ανθρώπινους ιστούς φυσιολογικούς και καρκινικούς. Μελέτες φασματομετρίας μάζας (ΜALDI-TOF MS/MS), όπου χρησιμοποιήθηκαν συνθετικά πεπτίδια και μελετήθηκε η αλληλεπίδραση τους με το άλας τριφθοροoξικού ψευδαργύρου έδειξαν ότι ο ψευδάργυρος δεσμεύεται τουλάχιστον από ένα αμινοξύ, και συγκεκριμένα μια κυστεΐνη ή μια ιστιδίνη, με πιθανή επίσης την ύπαρξη ενός συμπλόκου κυστεΐνης-ψευδαραργύρου-ιστιδίνης. Περαιτέρω ερευνητική εργασία απαιτείται για να διευκρινιστεί ο ακριβής χημικός μηχανισμός της μονιμοποίησης με ψευδάργυρο. Τελικά, αυτό το νέο, μη τοξικό και οικονομικό μονιμοποιητικό ιστών και κυττάρων θα μπορούσε να χρησιμοποιηθεί σε τακτική βάση τόσο σε ερευνητικά όσο και σε διαγνωστικά εργαστήρια και επιπλέον να επιτρέψει γενομικές και πρωτεομικές μελέτες

A PFA fixation step is an essential requirement for successful NoRISH.

<p>Summary of the results of <i>in situ</i> hybridization experiments on sections of E13.5 mouse embryos with the <i>Lhx6</i> probe using several combinations of fixatives.</p><p>Signal intensity was evaluated on a four-point scale, with 1 being weak, and 4 being very strong. -: no signal.</p

The morphology of Z7-fixed tissues is comparable to that of PFA-fixed tissues.

<p>Histological assessment of the morphology of the sections of Z7-fixed and PFA-fixed embryos following H&E staining. Tissue morphology was evaluated on a four-point scale, with 1 being poor, and 4 being excellent.</p

Tissue fixation in Z7 improves the sensitivity of NoRISH.

<p>Summary of the results of <i>in situ</i> hybridization experiments on sections of mouse embryos with <i>Lhx6</i>, <i>Lhx7</i>, <i>ret</i> and <i>ncapg</i> antisense probes. Signal intensity was evaluated on a four-point scale, with 1 being weak, and 4 being very strong. -: no signal.</p>*<p>depending on the stage.</p

The introduction of Z7 improves the sensitivity of NoRISH.

<p><i>In situ</i> hybridization on sections of E13.5 mouse embryo head fixed with Z7 (A–F) or PFA (G–I) and hybridized with a sense (F, I) or an antisense RNA probe of the <i>Lhx6</i> gene (A–E, G,H). A: Z7 1 h, B: Z7 3 h, C: Z7 6 h, D: Z7 12 h, E: Z7 24 h, F: Z7 1 h, G–I: PFA 24 h. Detection time: 3 h (A–F), 6 h (G–I). mge: medial ganglionic eminence, hy: hypothalamus. Scale bar: 250 µm.</p