Quantification of mRNA in single cells and modelling of RT-qPCR induced noise

Low concentration of guanidine thiocyanate was used to fully lyse single pancreatic β-cells followed by RT-qPCR without the need for purification. The accuracy of the measurements was determined by a quantitative noise-model of the reverse transcription and PCR. The noise is insignificant for initial copy numbers >100 while at lower copy numbers the noise intrinsic of the PCR increases sharply, eventually obscuring quantitative measurements. Importantly, the model allows us to determine the RT efficiency without using artificial RNA as a standard. The experimental setup was applied on single endocrine cells, where the technical and biological noise levels were determined.Noise in single-cell RT-qPCR is insignificant compared to biological cell-to-cell variation in mRNA levels for medium and high abundance transcripts. To minimize the technical noise in single-cell RT-qPCR, the mRNA should be analyzed with a single RT reaction, and a single qPCR reaction per gene.Cells in a population are in many aspects unique in their characteristics, even in a seemingly homogenous culture or tissue. Single cell gene expression levels–protein as well as mRNA–show large cell-to-cell variations both in a resting state and when exposed to stimuli, stemming in part from the stochastic nature of gene expression [1-6]. This implies that data obtained from a population of cells cannot be assumed to reflect the behaviour of the individual cell. Instead, cells must be assayed one at a time which, in the case of mRNA measurements, requires characterization of femtograms of mRNA [7].Quantitative reverse transcription polymerase chain reaction (RT-qPCR) offers sufficient sensitivity and it is the gold standard for mRNA quantification [8,9]. Coupled with a method to handle and collect individual cells, quantification of mRNA in single cells is feasible for most laboratories today. Previously published protocols for single-cell RT-PCR are generally non-quantitative and specialized for a particul