Precision in RNA molecular measurement

Sanders, Rebecca 2016. Precision in RNA molecular measurement. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Measurement of gene expression profiles represents a snapshot of cellular metabolism or activity at the molecular scale. This involves measurement of messenger (m)RNA employing techniques such as reverse transcription quantitative polymerase chain reaction (RT-qPCR). To truly assign biological significance to associated findings, researchers must consider the idiosyncrasies of this method and associated technical error, termed measurement uncertainty. Significant error can occur at sample source, RNA extraction, RT and qPCR levels. This thesis explores the steps which may introduce potential bias. It is hypothesised that error in mRNA measurement can be partitioned across different experimental stages. Within this thesis, RNA measurement from sample source to qPCR has been analysed at each stage to delineate variability contributions attributed to specific steps using synthetic and validated endogenous reference genes, single cell lines, 3D models and complex bone tissue. These data determined that total RNA yields remained consistent between treatment (2D cell mineralisation, 3D co-culture mechanical loading) and control groups (p > 0.06). Sample complexity was positively correlated with RNA extraction yield variability. Evaluation of different extraction methods demonstrated that total RNA yields differed between methods (p < 0.001). Assessing total RNA quantity and quality, different metrics (Bioanalyzer, Nanodrop and Qubit) generated different yield estimates (p < 0.05), although quality estimates from different metrics were found to be comparable. In addition, different cell batches (cultures of the same cells from different cryo vials) generated disparate total RNA yields (p < 0.02), with variable quality estimates, despite normalisation for cell count. RT-digital PCR analysis revealed quantification differences and detection sensitivity biases between different RT enzymes (p < 0.0001), suggesting cDNA prepared using different RT enzymes cannot be meaningfully compared. The ERCC synthetic targets were variable under the model conditions assessed and therefore not suitable as normalisers in these circumstances. This work provides a guide for the approaches necessary to reduce error, improve experimental design and minimise uncertainties.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > QR Microbiology
Uncontrolled Keywords:	mRNA, measurement uncertainty, precision, RT-qPCR, variability, standardisation, MIQE
Date of First Compliant Deposit:	27 May 2016
Date of Acceptance:	25 May 0206
Last Modified:	18 Aug 2021 12:58
URI:	https://orca.cardiff.ac.uk/id/eprint/91295

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