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

Bengtsson M, Hemberg M, Rorsman P, Ståhlberg A - BMC Mol. Biol. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, The Churchill Hospital, Oxford, OX3 7LJ, UK. martin.bengtsson@med.lu.se

ABSTRACT

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.

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Related in: MedlinePlus

Evaluation of lysis buffers. (A) Determination of lysis efficiency. Each bar indicate relative yield of Ins2 using a single pancreatic islet (~1000 cells) as starting material. Each islet was treated with indicated concentrations of either NP-40, with and without proteinase K (Prot K) treatment, or guanidine thiocyanate (GuSCN). Only lysis with 0.5 M GuSCN had a significant effect (>500-fold increase) compared to the water control (p < 0.001, n = 3). The value of the control was arbitrarily set to 1. Similar results were obtained for Gcg and Rps29. (B) Effect of lysis buffers on RT reaction yield. Identical amounts of purified islet total RNA was used as starting material. Relative yields of five genes were analysed: Ins1, Gcg, Sst, Gapdh and Rps29. Increasing concentrations of GuSCN was added to the RT reaction. There is a significant difference for all genes between control and both 40 mM and 120 mM (p < 0.05) but not 80 mM. The expression value was arbitrarily set to 1 for all genes at 0 mM GuSCN. Values are mean ± SEM for three separate experiments. The experiments in (A) and (B) were carried out without the presence of RNase inhibitor.
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Figure 2: Evaluation of lysis buffers. (A) Determination of lysis efficiency. Each bar indicate relative yield of Ins2 using a single pancreatic islet (~1000 cells) as starting material. Each islet was treated with indicated concentrations of either NP-40, with and without proteinase K (Prot K) treatment, or guanidine thiocyanate (GuSCN). Only lysis with 0.5 M GuSCN had a significant effect (>500-fold increase) compared to the water control (p < 0.001, n = 3). The value of the control was arbitrarily set to 1. Similar results were obtained for Gcg and Rps29. (B) Effect of lysis buffers on RT reaction yield. Identical amounts of purified islet total RNA was used as starting material. Relative yields of five genes were analysed: Ins1, Gcg, Sst, Gapdh and Rps29. Increasing concentrations of GuSCN was added to the RT reaction. There is a significant difference for all genes between control and both 40 mM and 120 mM (p < 0.05) but not 80 mM. The expression value was arbitrarily set to 1 for all genes at 0 mM GuSCN. Values are mean ± SEM for three separate experiments. The experiments in (A) and (B) were carried out without the presence of RNase inhibitor.

Mentions: The process of single-cell RT-qPCR is summarized in Figure 1. Our approach, which does not involve mRNA purification, requires a lysis solution that is compatible with downstream enzymatic reactions. In addition, it should 1) disrupt the cell membrane; 2) make the mRNA accessible for reverse transcription; and 3) maintain mRNA integrity. Two agents were evaluated for this task: NP-40, a weak, non-chaotropic detergent (also referred to as Igepal CA-630) and guanidine thiocyanate (GuSCN) a strong, chaotropic compound. Five different lysis conditions were tested in terms of their ability to lyse one cell cluster (pancreatic islet of Langerhans, each containing ~1000 cells), assessed by the amount of accessible insulin II (Ins2) transcripts using RT-qPCR (Figure 2A). NP-40 had no effect compared to control (water) when used at concentrations of 0.5% or 4%, indicating that these lysis conditions are too weak to dissociate the islet and lyse the cells. Proteinase K is commonly used in lysis protocols, but had no beneficial effect when added in the presence of 0.5% NP-40. GuSCN based lysis buffer provided efficient lysis of the islet using a concentration of 0.5 M and increased the RNA yield 600-fold compared to control conditions (Figure 2A). Lysis of a compact cluster of cells is clearly more challenging than dissociated cells. We evaluated the latter using another cell type, primary astrocytes, and the RNA yield was here within a 2.5-fold range for both 0.5 M GuSCN and 0.5% NP-40 compared to the water control (Additional file 1, Figure 1). This indicates that any of the tested lysis conditions will suffice for complete cell lysis of dissociated cells, while 0.5 M GuSCN is required to break apart cell clusters. In a sample containing a single lysed cell, the RNA is assumed to be evenly distributed in the solution. We tested this hypothesis by splitting vortexed single-cell lysates into three separate RT-reactions, followed by qPCR using Ins2 primers. Samples lysed in 0.5 M GuSCN showed 80% lower intra-assay variation than cells emptied in 0.5% NP-40, suggesting that GuSCN lyses the cell and efficiently homogenates the mRNA (Additional file 1: Figure 2A).


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

Bengtsson M, Hemberg M, Rorsman P, Ståhlberg A - BMC Mol. Biol. (2008)

Evaluation of lysis buffers. (A) Determination of lysis efficiency. Each bar indicate relative yield of Ins2 using a single pancreatic islet (~1000 cells) as starting material. Each islet was treated with indicated concentrations of either NP-40, with and without proteinase K (Prot K) treatment, or guanidine thiocyanate (GuSCN). Only lysis with 0.5 M GuSCN had a significant effect (>500-fold increase) compared to the water control (p < 0.001, n = 3). The value of the control was arbitrarily set to 1. Similar results were obtained for Gcg and Rps29. (B) Effect of lysis buffers on RT reaction yield. Identical amounts of purified islet total RNA was used as starting material. Relative yields of five genes were analysed: Ins1, Gcg, Sst, Gapdh and Rps29. Increasing concentrations of GuSCN was added to the RT reaction. There is a significant difference for all genes between control and both 40 mM and 120 mM (p < 0.05) but not 80 mM. The expression value was arbitrarily set to 1 for all genes at 0 mM GuSCN. Values are mean ± SEM for three separate experiments. The experiments in (A) and (B) were carried out without the presence of RNase inhibitor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Evaluation of lysis buffers. (A) Determination of lysis efficiency. Each bar indicate relative yield of Ins2 using a single pancreatic islet (~1000 cells) as starting material. Each islet was treated with indicated concentrations of either NP-40, with and without proteinase K (Prot K) treatment, or guanidine thiocyanate (GuSCN). Only lysis with 0.5 M GuSCN had a significant effect (>500-fold increase) compared to the water control (p < 0.001, n = 3). The value of the control was arbitrarily set to 1. Similar results were obtained for Gcg and Rps29. (B) Effect of lysis buffers on RT reaction yield. Identical amounts of purified islet total RNA was used as starting material. Relative yields of five genes were analysed: Ins1, Gcg, Sst, Gapdh and Rps29. Increasing concentrations of GuSCN was added to the RT reaction. There is a significant difference for all genes between control and both 40 mM and 120 mM (p < 0.05) but not 80 mM. The expression value was arbitrarily set to 1 for all genes at 0 mM GuSCN. Values are mean ± SEM for three separate experiments. The experiments in (A) and (B) were carried out without the presence of RNase inhibitor.
Mentions: The process of single-cell RT-qPCR is summarized in Figure 1. Our approach, which does not involve mRNA purification, requires a lysis solution that is compatible with downstream enzymatic reactions. In addition, it should 1) disrupt the cell membrane; 2) make the mRNA accessible for reverse transcription; and 3) maintain mRNA integrity. Two agents were evaluated for this task: NP-40, a weak, non-chaotropic detergent (also referred to as Igepal CA-630) and guanidine thiocyanate (GuSCN) a strong, chaotropic compound. Five different lysis conditions were tested in terms of their ability to lyse one cell cluster (pancreatic islet of Langerhans, each containing ~1000 cells), assessed by the amount of accessible insulin II (Ins2) transcripts using RT-qPCR (Figure 2A). NP-40 had no effect compared to control (water) when used at concentrations of 0.5% or 4%, indicating that these lysis conditions are too weak to dissociate the islet and lyse the cells. Proteinase K is commonly used in lysis protocols, but had no beneficial effect when added in the presence of 0.5% NP-40. GuSCN based lysis buffer provided efficient lysis of the islet using a concentration of 0.5 M and increased the RNA yield 600-fold compared to control conditions (Figure 2A). Lysis of a compact cluster of cells is clearly more challenging than dissociated cells. We evaluated the latter using another cell type, primary astrocytes, and the RNA yield was here within a 2.5-fold range for both 0.5 M GuSCN and 0.5% NP-40 compared to the water control (Additional file 1, Figure 1). This indicates that any of the tested lysis conditions will suffice for complete cell lysis of dissociated cells, while 0.5 M GuSCN is required to break apart cell clusters. In a sample containing a single lysed cell, the RNA is assumed to be evenly distributed in the solution. We tested this hypothesis by splitting vortexed single-cell lysates into three separate RT-reactions, followed by qPCR using Ins2 primers. Samples lysed in 0.5 M GuSCN showed 80% lower intra-assay variation than cells emptied in 0.5% NP-40, suggesting that GuSCN lyses the cell and efficiently homogenates the mRNA (Additional file 1: Figure 2A).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, The Churchill Hospital, Oxford, OX3 7LJ, UK. martin.bengtsson@med.lu.se

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus