The dynamic changes of megakaryocyte transcriptome during differentiation
Malin, Aino (2026)
2026
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2026-04-17
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202604163962
https://urn.fi/URN:NBN:fi:tuni-202604163962
Tiivistelmä
Megakaryocyte cells are large and rare hematopoietic cells functioning primarily in producing platelets. They undergo a complex maturation process called megakaryopoiesis in which they grow in size and multiply their genomic content without cell division, becoming polyploid. Platelets are anuclear and get their cytoplasmic contents from the parental cell. Megakaryocyte maturation provides an interesting platform for studying transcription dynamics during a process where the cellular environment undergoes drastic change. Nascent RNA refers to RNA that is newly synthe-sised and it is important in understanding transcription dynamics. The aims of the thesis were to optimise nascent RNA labelling and extraction protocol study transcription dynamics during megakaryocyte maturation.
The main method used in the thesis is pulse labelling of megakaryocyte nascent RNA with 5-ethynyl-uridine (5EU) and 4-thio-uridine (4SU), which are nucleoside analogs commonly used in metabolic labelling experiments. Fluorescent imaging was conducted to visualise the nascent RNA and to confirm successful labelling. Streptavidin pulldown was conducted to quantify the nascent RNA pulldown efficiency and nascent RNA levels between labelling times and maturation stages of the cells. Additionally, quality control methods were used to study the integrity of the isolated RNA in different steps of the protocol.
Fluorescent imaging showed that 5EU is taken up by the cells and incorporated to nascent RNA. On the contrary, 4SU label caused significant background signal in imaging experiments, hence resulting in inconclusive results. Pulldown experiment was successful with 5EU and pro-vided information about transcription levels at different maturation stages. Proliferative cells were most transcriptionally active followed by early stage cells and late stage cells. However, these analysis of efficacy still need validation with more replicates of samples.
In conclusion, the main aim of the thesis was achieved since the method of 5EU labelling and enriching nascent RNA was successfully optimised. The limitations of the thesis are that the protocol is laborious and requires a lot of starting material, because the efficiency of the pulldown is low. In the future, sequencing nascent RNA will be done to obtain knowledge about the tran-scriptional dynamics during megakaryocyte maturation. This thesis provides a starting point for further research.
The main method used in the thesis is pulse labelling of megakaryocyte nascent RNA with 5-ethynyl-uridine (5EU) and 4-thio-uridine (4SU), which are nucleoside analogs commonly used in metabolic labelling experiments. Fluorescent imaging was conducted to visualise the nascent RNA and to confirm successful labelling. Streptavidin pulldown was conducted to quantify the nascent RNA pulldown efficiency and nascent RNA levels between labelling times and maturation stages of the cells. Additionally, quality control methods were used to study the integrity of the isolated RNA in different steps of the protocol.
Fluorescent imaging showed that 5EU is taken up by the cells and incorporated to nascent RNA. On the contrary, 4SU label caused significant background signal in imaging experiments, hence resulting in inconclusive results. Pulldown experiment was successful with 5EU and pro-vided information about transcription levels at different maturation stages. Proliferative cells were most transcriptionally active followed by early stage cells and late stage cells. However, these analysis of efficacy still need validation with more replicates of samples.
In conclusion, the main aim of the thesis was achieved since the method of 5EU labelling and enriching nascent RNA was successfully optimised. The limitations of the thesis are that the protocol is laborious and requires a lot of starting material, because the efficiency of the pulldown is low. In the future, sequencing nascent RNA will be done to obtain knowledge about the tran-scriptional dynamics during megakaryocyte maturation. This thesis provides a starting point for further research.