Flemming_2011_direct.pdf (4.67 MB)
Direct amplification of nucleic acids from cells
thesisposted on 2022-11-18, 14:49 authored by Paul Fleming
All biological conditions, whether pathological or a cellular response to extra cellular signals, are reflected in changes in gene expression. Gene expression profiling is therefore a valuable tool in many areas, including clinical diagnostics. qPCR has become the gold standard of gene expression profiling due to its range and sensitivity. Continuous-flow PCR devices hold numerous advantages over stationary well based systems, the foremost being their high throughput capabilities. A number of successful continuous flow microfluidic devices capable of real time and end point detection of gene expression have been documented. The majority of these devices use cDNA as their template, with reverse transcription carried out in the laboratory prior to loading onto the device. The RNA extraction step can also be time consuming and labour intensive. Successfully integrating these preparatory steps into the device design can reduce sample crossover and contamination and hands on time required. A high-throughput continuous flow instrument capable of performing qPCR using microfluidic technology was designed by Stokes Institute. The objective of this research thesis is to integrate the preparatory steps in the gene expression workflow succeeding the PCR step. These steps include cell encapsulation, cell lysis, the addition of the reagents required for RT-PCR, and the reverse transcription reaction. To achieve this objective, two thermal modules were designed and added to the device upstream of the PCR module. The first of these was a cell lysis module, to rupture the cells and create a cell lysate, and the second was a reverse transcription module which created complementary DNA which is a suitable template for the PCR. Sets of droplets containing the cell lysis buffer, RT-PCR components and the cells themselves were created through dipping. In addition, mixing steps were added to mix the cell lysis buffer with the cell suspension and to add the RT-PCR components to the cell lysate. An end-point detection system was incorporated into the system which allowed amplified droplets to be distinguished from un-amplified droplets. Successful one-step RT-PCR was demonstrated in 300 nl flowing droplets from both isolated RNA samples and cell lysates. Encapsulation of the reaction droplets allowed the elimination of any contamination which was not detected. Amplification was detected from as low as 1x10-5 ng RNA/μl for isolated RNA and from 1 cell equivalent/droplet from cell lysates. The addition of a mixing step to add the RT-PCR components further reduced sample preparation time. On-device cell lysis was also shown through the encapsulation of REH cells and the addition of a cell lysis buffer through a mixing step before moving the droplets through a cell lysis zone. The resultant cell lysates were shown to amplify on a commercially available benchtop thermal cycler. The combination of removing the labour intensive RNA extraction step, the addition of reaction components, performing one-step reverse-transcription PCR and fluorescence detection on the device helps moves this technology towards a cells-tosignal system. To the author's knowledge, this is the first continuous flow biphasic system capable of performing one-step RT-PCR from cell lysates, with an integrated detection setup and the automated addition of the RT-PCR components.
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- Faculty of Science and Engineering
First supervisorDalton, Tara
Other Funding informationIRC
Department or School
- School of Engineering