Development of a method for the analysis of serglycin proteoglycan gene expression in canine blood samples
Cancer is a highly prevalent disease among canine breeds. Nowadays, mainly histology parameters are used to determine the prognosis and for selecting treatment strategies. Due to the high degree of morbidity and mortality in cancer affected dogs, there is a strong need to identify additional dia...
| Autor principal: | |
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| Formato: | First cycle, G2E |
| Lenguaje: | sueco Inglés |
| Publicado: |
2018
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| Materias: | |
| Acceso en línea: | https://stud.epsilon.slu.se/13860/ |
| _version_ | 1855572313687195648 |
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| author | Löfqvist, Kristin |
| author_browse | Löfqvist, Kristin |
| author_facet | Löfqvist, Kristin |
| author_sort | Löfqvist, Kristin |
| collection | Epsilon Archive for Student Projects |
| description | Cancer is a highly prevalent disease among canine breeds.
Nowadays, mainly histology parameters are used to determine the
prognosis and for selecting treatment strategies.
Due to the high degree of morbidity and mortality in cancer affected
dogs, there is a strong need to identify additional diagnostic methods
and biomarkers.
Serglycin is an intracellular proteoglycan found to be overexpressed
in several types of cancer, including aggressive canine mammary
cancer. High expression levels of serglycin in cancer cells are con-
sidered to correlate with cancer metastases and a poor prognosis for
the patient. Detection of serglycin expression may, therefore, be a
potential diagnostic marker for metastatic cancer in canine breeds.
A known molecular technology used daily in medical diagnostics is
quantitative polymerase chain reaction (qPCR). QPCR is primarily
used to identify the expression pattern of specific genes. The aim of
this study was to establish a qPCR assay that could be used for de-
tection of serglycin expression in canine blood. The focus of the
study was to validate potential reference genes as well as optimiza-
tion of a functional qPCR assay. For this purpose, blood samples
from canine donors with unknown disease history were chosen.
Total RNA from each blood sample was extracted it an optimized
TRIzol protocol. The samples were first tested in a step-down PCR,
and then further tested in three different qPCR optimization steps.
Both the step-down PCR and the qPCR included four genes;; SRGN
and three references genes EEF2, HPRT and ACTIN B. The qPCR
assays showed high specificity and sensitivity for two of the genes,
SRGN and EEF2.Primer pairs for each of the two genes showed
efficiency values within the range of 90 % to 105%, and their reflec-
tion of linearity was R2>0.980. The optimal annealing temperature for
the SRGN and EEF2 gene was set as 62 °C with a 300 nM primer
concentration. Unfortunately, the published primer-design for two of
the reference genes, i.e. HPRT and ACTIN B were poorly designed
resulted in amplification of unwanted DNA and primer dimer for-
mation.
In conclusion, the presented method in this study showed evidence
that serglycin could be detected in small amounts of canine blood
utilizing the described qPCR assay. We concluded that the EEF2
gene is a stable reference gene for studying gene expression in dog
blood with qPCR. However, in order to be able to apply our method
in further studies, additional suitable reference genes need to be
identified, tested and further validated. |
| format | First cycle, G2E |
| id | RepoSLU13860 |
| institution | Swedish University of Agricultural Sciences |
| language | Swedish Inglés |
| publishDate | 2018 |
| publishDateSort | 2018 |
| record_format | eprints |
| spelling | RepoSLU138602020-05-20T10:53:33Z https://stud.epsilon.slu.se/13860/ Development of a method for the analysis of serglycin proteoglycan gene expression in canine blood samples Löfqvist, Kristin Animal diseases Cancer is a highly prevalent disease among canine breeds. Nowadays, mainly histology parameters are used to determine the prognosis and for selecting treatment strategies. Due to the high degree of morbidity and mortality in cancer affected dogs, there is a strong need to identify additional diagnostic methods and biomarkers. Serglycin is an intracellular proteoglycan found to be overexpressed in several types of cancer, including aggressive canine mammary cancer. High expression levels of serglycin in cancer cells are con- sidered to correlate with cancer metastases and a poor prognosis for the patient. Detection of serglycin expression may, therefore, be a potential diagnostic marker for metastatic cancer in canine breeds. A known molecular technology used daily in medical diagnostics is quantitative polymerase chain reaction (qPCR). QPCR is primarily used to identify the expression pattern of specific genes. The aim of this study was to establish a qPCR assay that could be used for de- tection of serglycin expression in canine blood. The focus of the study was to validate potential reference genes as well as optimiza- tion of a functional qPCR assay. For this purpose, blood samples from canine donors with unknown disease history were chosen. Total RNA from each blood sample was extracted it an optimized TRIzol protocol. The samples were first tested in a step-down PCR, and then further tested in three different qPCR optimization steps. Both the step-down PCR and the qPCR included four genes;; SRGN and three references genes EEF2, HPRT and ACTIN B. The qPCR assays showed high specificity and sensitivity for two of the genes, SRGN and EEF2.Primer pairs for each of the two genes showed efficiency values within the range of 90 % to 105%, and their reflec- tion of linearity was R2>0.980. The optimal annealing temperature for the SRGN and EEF2 gene was set as 62 °C with a 300 nM primer concentration. Unfortunately, the published primer-design for two of the reference genes, i.e. HPRT and ACTIN B were poorly designed resulted in amplification of unwanted DNA and primer dimer for- mation. In conclusion, the presented method in this study showed evidence that serglycin could be detected in small amounts of canine blood utilizing the described qPCR assay. We concluded that the EEF2 gene is a stable reference gene for studying gene expression in dog blood with qPCR. However, in order to be able to apply our method in further studies, additional suitable reference genes need to be identified, tested and further validated. Elakartad cancer förkommer med hög frekvens bland många hund- raser. Olika histologiska och histopatologiska parametrar används huvudsakligen idag för att bestämma prognos och välja behandlings- strategi. På grund av den höga graden av sjuklighet och mortalitet inom elakartad cancer hos hund, behövs ytterligare diagnostiska metoder och biomarkörer behöver utvärderas för användning på kli- niken. Serglycin är en intracellulär proteoglykan som har visat sig vara överuttryckt i flera typer av cancer, inklusive aggressiva juver- tumörer hos hund. Höga expressionsnivåer av serglycin i cancercel- ler anses vara korrelerade med cancermetastaser och en dålig pro- gnos för patienten. Detektion av serglycin-uttryck kan därmed vara en potentiell diagnostisk metod för att bedöma risk för cancer- metastaser hos olika hundraser. En känd molekylärteknik som an- vänds dagligen inom medicinsk diagnostik är kvantitativ polymeras kedjereaktion (qPCR). qPCR används främst för att identifiera ut- trycksmönstret av specifika gener. Syftet med studien var att etablera en qPCR-analysmetod för att kunna detektera serglycin- (SRGN-) uttryck i hundblod, med målet att metoden sedan ska kunna använ- das för prognostisk screening av hundpatienter. Studiens fokus lades vid validering av potentiella referensgener samt optimering av en funktionell qPCR-analys. För detta ändamål valdes blodprover från hundar med okänd sjukdomshistoria. Totala mängden RNA isolera- des med hjälp av ett eget designat TRIzol-protokoll. Först testades proverna i en step-down PCR för att sedan testats vidare i totalt tre olika qPCR optimeringsanalyser. Både step-down PCR och qPCR inkluderade fyra gener;; SRGN och tre referensgener EEF2, HPRT och ACTIN B. qPCR-analysen visade sig ha hög specificitet och känslighet för två av generna SRGN och EEF2. Primerparen för var och en av de två generna visade sig ha effektivitesvärden inom inter- vallet 90% till 105% och deras R2 värden låg över 0,980. Den opti- mala annealing-temperaturen för SRGN och EEF2 generna sattes till 62°C med 300 nM primerkoncentration. Tyvärr, den publicerade pri- mer-designen för två av de valda referensgenerna HPRT och ACTIN B visade sig vara sämre vilket ledde till amplifiering av oönskat DNA och bildning av primerdimerer. Som slutsats, metodutvecklingen i denna studie visade att serglycin-uttrycket kunde detekteras i små volymer av hundblod med hjälp av qPCR-analys, samt att EEF2 är en stabil referensgen för att kvantifiera genuttryck i hundblod med qPCR. Men för att kunna tillämpa metoden i fortsatta studier behöver ytterligare lämpliga referensgener identifieras och valideras. 2018-10-12 First cycle, G2E NonPeerReviewed application/pdf sv https://stud.epsilon.slu.se/13860/7/lofqvist_k_080618.pdf Löfqvist, Kristin, 2018. Development of a method for the analysis of serglycin proteoglycan gene expression in canine blood samples. First cycle, G2E. Uppsala: (VH) > Dept. of Biomedical Sciences and Veterinary Public Health (until 231231) <https://stud.epsilon.slu.se/view/divisions/OID-713.html> urn:nbn:se:slu:epsilon-s-9889 eng |
| spellingShingle | Animal diseases Löfqvist, Kristin Development of a method for the analysis of serglycin proteoglycan gene expression in canine blood samples |
| title | Development of a method for the analysis of serglycin proteoglycan gene expression in canine blood samples |
| title_full | Development of a method for the analysis of serglycin proteoglycan gene expression in canine blood samples |
| title_fullStr | Development of a method for the analysis of serglycin proteoglycan gene expression in canine blood samples |
| title_full_unstemmed | Development of a method for the analysis of serglycin proteoglycan gene expression in canine blood samples |
| title_short | Development of a method for the analysis of serglycin proteoglycan gene expression in canine blood samples |
| title_sort | development of a method for the analysis of serglycin proteoglycan gene expression in canine blood samples |
| topic | Animal diseases |
| url | https://stud.epsilon.slu.se/13860/ https://stud.epsilon.slu.se/13860/ |