BACKGROUND Gene expression has a strong stochastic element resulting in highly variable mRNA levels between individual cells, even in a seemingly homogeneous cell population. Access to fundamental information about cellular mechanisms, such as correlated gene expression, motivates measurements of multiple genes in individual cells. Quantitative reverse transcription PCR (RT-qPCR) is the most accessible method which provides sufficiently accurate measurements of mRNA in single cells. RESULTS Low concentration of guanidine thiocyanate was used to fully lyse single pancreatic beta-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. CONCLUSION 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.

中文翻译：

---

单细胞中 mRNA 的定量和 RT-qPCR 诱导噪声的建模。

背景基因表达具有强随机因素，导致单个细胞之间的mRNA水平高度可变，即使在看似同质的细胞群中也是如此。获取有关细胞机制的基本信息，例如相关基因表达，激发了对单个细胞中多个基因的测量。定量逆转录 PCR (RT-qPCR) 是最容易获得的方法，它可以提供足够准确的单细胞 mRNA 测量值。结果 低浓度的硫氰酸胍用于完全裂解单个胰腺 β 细胞，然后进行 RT-qPCR，无需纯化。测量的准确性由逆转录和 PCR 的定量噪声模型确定。噪声对于初始拷贝数来说是微不足道的 > 100 而在较低拷贝数下，PCR 固有的噪声急剧增加，最终使定量测量变得模糊。重要的是，该模型允许我们在不使用人工 RNA 作为标准的情况下确定 RT 效率。实验装置应用于单个内分泌细胞，确定技术和生物噪声水平。结论 与中等和高丰度转录本 mRNA 水平的生物细胞间变异相比，单细胞 RT-qPCR 中的噪声微不足道。为了最大限度地减少单细胞 RT-qPCR 中的技术噪音，应使用单个 RT 反应和每个基因的单个 qPCR 反应分析 mRNA。该模型允许我们在不使用人工 RNA 作为标准的情况下确定 RT 效率。实验装置应用于单个内分泌细胞，确定技术和生物噪声水平。结论 与中等和高丰度转录本 mRNA 水平的生物细胞间变异相比，单细胞 RT-qPCR 中的噪声微不足道。为了最大限度地减少单细胞 RT-qPCR 中的技术噪音，应使用单个 RT 反应和每个基因的单个 qPCR 反应分析 mRNA。该模型允许我们在不使用人工 RNA 作为标准的情况下确定 RT 效率。实验装置应用于单个内分泌细胞，确定技术和生物噪声水平。结论 与中等和高丰度转录本 mRNA 水平的生物细胞间变异相比，单细胞 RT-qPCR 中的噪声微不足道。为了最大限度地减少单细胞 RT-qPCR 中的技术噪音，应使用单个 RT 反应和每个基因的单个 qPCR 反应分析 mRNA。