Molekylär karakterisering av cirkulerande tumörceller i bröstcancer
|Koordinator||Göteborgs Universitet - Institutionen för biomedicin, avdelningen för patologi|
|Bidrag från Vinnova||2 121 400 kronor|
|Projektets löptid||augusti 2014 - mars 2018|
|Ansökningsomgång||VINNMER Marie Curie Incoming 2014-03-14|
Syfte och mål
The aim of this project was to improve single/rare cell analysis, especially in the field of circulating tumor cells (CTCs). Therefore, the labs from Graz (Austria) and Gothenburg (Sweden) teamed up with a Gilupi (Germany), a company experienced in in vivo enrichment of CTCs. The primary objective was to establish a method capable of characterizing cells at the single cell level beyond mere CTC enumeration. Therefore we aimed at implementing RNA analysis into single-cell workflow allowing targeting dynamic processes.
Resultat och förväntade effekter
We developed a protocol combining single-cell isolation using fluorescence activated cell sorting with global transcriptome amplification of single cells. This protocol allows multiple analyses from single cells as well as combining target-specific and screening-based analyses at the single cell level. Testing the method on 96 single cells allowed us to unbiasedly investigate the cell cycle. Apart from known genes we identified novel genes involved in cell cycle regulation and detected subpopulations within our G1 cells referring to early and late G1 phase.
Upplägg och genomförande
We first implemented our established single-cell lysis protocol into the RNA-sequencing library preparation workflow and run RNA whole transcriptome amplification (WTA) evaluating the performance of the WTA using high-throughput qPCR. This test confirmed the technical variation of our new method to be lower than the heterogeneity among single cells. Applied on 96 sorted single cells we performed cell cycle analysis: The amplified transcriptomes of the single cells were forwarded to RNA-sequencing and subjected comprehensive data analysis