BACKGROUND
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  <cit> .
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  <cit> .
quantitative reverse transcription polymerase chain reaction  offers sufficient sensitivity and it is the gold standard for mrna quantification  <cit> .
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 particular cell-type or involve laborious purification steps  <cit> .
for single-cell mrna quantification to become common practice, simple and generally applicable methods are needed.
indeed, non-proprietary protocols not requiring purification have been presented, but they involve only weak detergents serving as lysis agents that do not inhibit nucleases  <cit> .
lack of appropriate controls for single-cell measurements makes it difficult to guarantee mrna integrity during collection and handling of cells.
noise in rt-qpcr measurements increases with decreasing initial copy numbers  <cit>  and quantification of rare transcripts pushes the measurement to a point where it no longer can be considered quantitative; at best the presence of mrna will be detected.
the details of this phenomenon–including the relative contribution of rt and qpcr respectively–are poorly investigated  <cit> .
previous reports utilizing single-cell rt-qpcr have left these questions largely unanswered or with inconsistent results: qpcr induced variation ranging from ~8%  at  <dig> cdna copies  <cit>  down to <1% at two copies  <cit>  have been shown.
without knowledge of the distribution and impact of the technical noise, it is hard to assess the validity of the measurements.
in this study, we present a single-cell rt-qpcr protocol without purification, with maintained mrna integrity, high reproducibility and no inhibition of rt or pcr reactions.
in addition, we investigate the nature of noise in rt-qpcr and through mathematical modelling we determine the accuracy in measurements of single mouse endocrine cells.
our model predicts how the noise in rt and pcr varies with different expression levels, which will aid in planning and design of single-cell rt-qpcr measurements.
we conclude that the inherent variability of rt-qpcr is negligible compared to naturally occurring variation between cells for abundantly expressed genes, while uncertainties in the pcr may obfuscate measurements for rare transcripts.
RESULTS
derivation of a purification-free protocol
the process of single-cell rt-qpcr is summarized in figure  <dig>  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- <dig>  a weak, non-chaotropic detergent  and guanidine thiocyanate  a strong, chaotropic compound.
five different lysis conditions were tested in terms of their ability to lyse one cell cluster , assessed by the amount of accessible insulin ii  transcripts using rt-qpcr .
np- <dig> had no effect compared to control  when used at concentrations of  <dig> % 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  <dig> % np- <dig>  guscn based lysis buffer provided efficient lysis of the islet using a concentration of  <dig>  m and increased the rna yield 600-fold compared to control conditions .
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  <dig> -fold range for both  <dig>  m guscn and  <dig> % np- <dig> compared to the water control .
this indicates that any of the tested lysis conditions will suffice for complete cell lysis of dissociated cells, while  <dig>  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 ins <dig> primers.
samples lysed in  <dig>  m guscn showed 80% lower intra-assay variation than cells emptied in  <dig> % np- <dig>  suggesting that guscn lyses the cell and efficiently homogenates the mrna .
the presence of potential inhibitors and rnases from the tissue or culture medium may reduce the cdna yield.
we addressed this concern by adding a known amount of purified total islet rna to tubes containing one of the following: 1) extracellular solution ; 2) one cell in  <dig>  m guscn in water; or 3) ten cells in  <dig>  m guscn in water .
the amount of rna in the cells was negligible compared to the externally added rna.
the yield was unaffected by the extracellular solution and in fact increased in the presence of cells in guscn, a phenomenon investigated further .
as an additional control for inhibitors, we added an equal amount  of in vitro transcribed artificial rna, based on the cyclophilin e  gene, to the lysis solution.
ppie was measured in every single-cell sample and deviation >1% led to exclusion from further analysis .
in figure 2b, an equal amount of purified rna was reverse transcribed in the presence of  <dig>   <dig>   <dig> and  <dig> mm guscn, followed by qpcr using five different primer pairs.
the rt-reaction efficiency was significantly improved  for all tested genes  using  <dig> mm guscn.
by contrast,  <dig> mm guscn had no effect whereas  <dig> mm guscn was strongly inhibitory.
we also evaluated the addition of  <dig> – <dig> % 2-mercapto ethanol, but no effect on the rt yield, either alone or in concert with guscn, was observed .
formation of correct pcr-products was confirmed by agarose gel electrophoresis.
neither of the other two lysis conditions, np- <dig> or proteinase k, had any discernible effect on rt yield .
taken together, we chose to use ~ <dig>  m guscn in the single-cell lysis solution due to superior cell lysis ability and positive effect on the rt reaction at the concentration of  <dig> mm.
rt- and qpcr-induced noise
the total technical noise level of rt-qpcr was estimated by diluting purified total rna from four to six different concentrations , ~10- <dig> copies of ribosomal protein s <dig> , ~50- <dig> copies of sry-box containing gene  <dig> , nestin  and glial fibrillary acidic protein  and ~500-5· <dig> copies of ins <dig> transcripts).
for each dilution,  <dig> identical rt-qpcr reactions were run.
the mrna concentrations were chosen to primarily cover low-medium abundance transcripts, i.e.
chgb, gfap, nes, rps <dig> and sox <dig>  consequently, the concentration of the high abundance ins <dig> transcript is 50– <dig> times higher.
the qpcr-specific noise was calculated from  <dig> qpcr replicates  at five different concentrations of mouse cdna.
the measurements were combined to create a model of rt- and qpcr-induced noise, as well as errors introduced by the liquid handling.
the technical reproducibility of rt and qpcr is represented here by the noise strength , for different initial copy numbers.
assuming that the noise from each reaction is additive, we can write the total noise as η2total = η2dilution + η2rt+ η2pcr and by fitting this model to the data, we can quantify the contribution of each component.
η2dilution corresponds to poisson-type noise which arises when a sample with a low number of molecules is imperfectly diluted.
additional noise is introduced by the rt-reaction and the final term corresponds to the noise introduced by the qpcr step.
in additional file  <dig>  we derive functional forms for each component and we show how they can be assessed from replicate measurements.
single-cell rt-qpcr
using a glass capillary mounted on a micromanipulator, we collected pancreatic β-cells incubated in  <dig>   <dig>   <dig> and  <dig> mm glucose and emptied the intact cell in  <dig> μl  <dig>  m guscn.
following reverse transcription, we subjected each single-cell's cdna to qpcr using primers for rps <dig>  chgb and ins <dig> .
the median expression level across the different glucose concentrations for these three genes were  <dig>   <dig> and  <dig> mrna copies per cell respectively.
some cells failed to generate a signal .
transcript level heterogeneity is high and the distribution is skewed, which is in agreement with previous studies of eukaryotic cells  <cit> .
one consequence of this heterogeneity is that the top 10% of the cells with the highest ins <dig> expression account for 50% of the total ins <dig> mrna.
increasing glucose concentrations altered the median ins <dig> expression from  <dig> mrna copies at  <dig> mm glucose to  <dig>   <dig>  and  <dig> mrna copies for  <dig>   <dig> and  <dig> mm glucose, respectively .
the increase was mainly due to an increasing fraction of cells with very high expression levels.
glucose did not have an effect on rps <dig> or chgb.
how much of this variability stems from true, biological variation and how much is attributable to the rt-qpcr?
the rt-qpcr noise model applied on the single-cell data suggests that the rt-qpcr noise is ~ <dig> % of the total variation in the ins <dig> expression .
for rps <dig>  which has lower expression, the range is 10–50% and for chgb 75–200%.
thus, the decreasing accuracy of the rt-qpcr at lower copy numbers heavily influences measurements of low-abundance transcripts such as chgb.
moreover, the 95% confidence level shows that a single measurement is accurate to within a factor of two when the expression level is > <dig> transcripts .
we conclude that in the case of abundantly expressed genes, the distribution of the measurement errors is very narrow compared to the biological noise and the fluctuations introduced by the experimental procedure are negligible.
finally, we investigated pcr inhibition caused by overloading the reaction with cdna  <cit> .
we tried to mitigate the pcr inhibition by decreasing the ratio between reverse transcriptase and taq polymerase, and found that a ratio ≤  <dig>  was required to completely remove inhibitory effects.
at low concentrations of rna, decreasing the rt-enzyme concentration did not have a negative effect on rt-yield .
discussion
individual cells have transcript levels which vary significantly from the population mean  <cit> .
the biological significance of this heterogeneity is still not clearly understood and it is an active area of research today.
to characterize this cell-to-cell variation, high precision measurements of single cells are required.
technically, such measurements are still very challenging and we have presented an rt-qpcr protocol which should be accessible for most laboratories.
by using a purification-free protocol we are both simplifying single-cell measurements and eliminating rna losses associated with current available purification protocols.
high concentrations  of guanidine salts are commonly used in standard rna purification protocols, where it denatures nucleases rapidly and liberates nucleic acids from bound proteins  <cit> .
since guscn strongly inhibits downstream reactions, the rna is normally purified.
at lower concentrations of guscn the effect diminishes, but rnases are completely inhibited even at  <dig> m guscn  <cit>  and we show that the cell lysis ability remains strong at  <dig>  m. by ensuring sufficient dilution  of the guscn prior reverse transcription, the concentration is brought down to regions where it does not interfere with the enzymatic reactions.
indeed, we demonstrate that low concentrations  even had a favourable effect on the rt yield and reproducibility.
this effect may be a result from reduced secondary structures of the mrna  <cit> , enhanced enzyme activity  <cit> , and/or that guscn protects the mrna from degradation.
we have also developed a mathematical model which allows us to quantify the contributions from the rt and pcr steps to the technical noise.
the only parameter in our model which is not directly obtained from the experimental data is the rt efficiency.
the rt efficiency can be estimated experimentally by rt-qpcr using known amounts of synthetic rna mimicking the properties of the native mrna  <cit> .
however, producing full length mrnas is challenging and it is common to use a truncated rna molecule which may not be reverse transcribed with the same efficiency as the native mrna.
instead, we use our model to determine the rt efficiency indirectly; using non-linear regression we find the rt efficiency which best fits the data.
we find that this indirect method gives realistic results for chgb, rps <dig>  gfap, nes and sox <dig> and we believe that it could be a useful way of estimating the rt efficiency for a given gene.
given the limited amount of mrna in a single cell  <cit> , how should the rt and qpcr experiments be designed in order to minimize technical noise?
we find that the benefit of replicate rt reactions is insignificant, depending on the rt efficiency, and we recommend a single rt reaction using all available mrna .
in figure  <dig>  we illustrate different strategies to distribute the cdna in qpcr reactions.
high dilution allows more genes and replicates to be analyzed, but this is at the price of lower mrna copy numbers, thereby increasing the measurement noise.
figures 6a–b shows how the technical noise increases when the sample is diluted.
curves show technical noise levels for each sample when it is split in  <dig>   <dig>   <dig> or  <dig> equal parts between rt and pcr.
figures 6c–d show that additional dilution of the cdna to allow qpcr replicates is not a suitable approach to decrease the technical noise.
in particular, for ins <dig> , which has a low rt efficiency there is a slight increase of the technical noise when the number of replicates is increased.
in contrast, for rps <dig> with a high rt efficiency there is a slight reduction of the technical noise for low mrna numbers.
in summary, the single-cell mrna should be reverse transcribed in a single rt reaction followed by one qpcr reaction per gene.
it is advantageous to use more of the single-cell cdna for low-abundance genes and a smaller part for abundantly expressed genes.
these conclusions are only valid for low mrna levels.
however, loading large volumes of cdna to the qpcr may cause inhibition.
in an effort to reduce this effect, we confirm previous findings suggesting a lowered reverse transcriptase concentration and an increased taq enzyme concentration  <cit> .
an alternate solution is to precipitate the single-cell cdna in ethanol, thus utilizing a larger fraction of the total amount in the qpcr  <cit> .
yet another option is multiplex pre-amplification rt-pcr for 10– <dig> cycles, followed by singleplex qpcr on the resulting pool of pcr-products <cit> .
this could decrease technical noise levels, provided that the multiplex pcr is efficient, reproducible and unbiased.
our model of the technical noise suggests that most of the noise in the method stems from the qpcr, at least when the initial template copy number is low and the rt-efficiency is relatively high.
however, our analyses show that the rt-efficiencies vary significantly across genes  <cit>  and for low efficiencies, the rt reaction will constitute a larger proportion of the total noise.
compared to the biological cell-to-cell variation, the technical noise is insignificant down to ~ <dig> copies, but becomes considerable at < <dig> rna copies.
the model includes only three experimental factors; the rt-reaction, dilution of the sample and the qpcr while in reality there are many more steps contributing to the total noise.
thus, our estimates of the noise components should be considered upper bounds since they will most likely become smaller if additional factors are included in the model.
CONCLUSIONS
we found that the strong denaturant guanidine thiocyanate can serve as lysis agent and rnase inhibitor and improve reverse transcription yield in purification-free single-cell rt-qpcr.
our protocol allows fast and streamlined measurements of single-cell gene expression.
analysis of technical noise caused by the rt- and pcr-reaction showed that this noise is insignificant compared to biological cell-to-cell variation at mrna copy numbers >~ <dig>  below this level technical noise stemming from the pcr, which increases dramatically when initial copy number is <~ <dig> cdna copies, becomes significant.
in addition, to achieve a high technical reproducibility, genes should be analyzed with lowest possible dilution between the rt and qpcr.
that is, a single qpcr measurement should be performed for each gene, where the number of genes to be analyzed determines the total dilution factor between rt and pcr.

 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. 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. quantitative reverse transcription polymerase chain reaction offers sufficient sensitivity and it is the gold standard for mrna quantification. 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 particular cell-type or involve laborious purification steps. for single-cell mrna quantification to become common practice, simple and generally applicable methods are needed. indeed, non-proprietary protocols not requiring purification have been presented, but they involve only weak detergents serving as lysis agents that do not inhibit nucleases. lack of appropriate controls for single-cell measurements makes it difficult to guarantee mrna integrity during collection and handling of cells. noise in rt-qpcr measurements increases with decreasing initial copy numbers and quantification of rare transcripts pushes the measurement to a point where it no longer can be considered quantitative; at best the presence of mrna will be detected. the details of this phenomenon–including the relative contribution of rt and qpcr respectively–are poorly investigated. previous reports utilizing single-cell rt-qpcr have left these questions largely unanswered or with inconsistent results: qpcr induced variation ranging from ~8% at cdna copies down to <1% at two copies have been shown. without knowledge of the distribution and impact of the technical noise, it is hard to assess the validity of the measurements. in this study, we present a single-cell rt-qpcr protocol without purification, with maintained mrna integrity, high reproducibility and no inhibition of rt or pcr reactions. in addition, we investigate the nature of noise in rt-qpcr and through mathematical modelling we determine the accuracy in measurements of single mouse endocrine cells. our model predicts how the noise in rt and pcr varies with different expression levels, which will aid in planning and design of single-cell rt-qpcr measurements. we conclude that the inherent variability of rt-qpcr is negligible compared to naturally occurring variation between cells for abundantly expressed genes, while uncertainties in the pcr may obfuscate measurements for rare transcripts.  derivation of a purification-free protocol the process of single-cell rt-qpcr is summarized in figure 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- a weak, non-chaotropic detergent and guanidine thiocyanate a strong, chaotropic compound. five different lysis conditions were tested in terms of their ability to lyse one cell cluster, assessed by the amount of accessible insulin ii transcripts using rt-qpcr. np- had no effect compared to control when used at concentrations of % 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 % np- guscn based lysis buffer provided efficient lysis of the islet using a concentration of m and increased the rna yield 600-fold compared to control conditions. 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 -fold range for both m guscn and % np- compared to the water control. this indicates that any of the tested lysis conditions will suffice for complete cell lysis of dissociated cells, while 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 ins primers. samples lysed in m guscn showed 80% lower intra-assay variation than cells emptied in % np- suggesting that guscn lyses the cell and efficiently homogenates the mrna. the presence of potential inhibitors and rnases from the tissue or culture medium may reduce the cdna yield. we addressed this concern by adding a known amount of purified total islet rna to tubes containing one of the following: 1) extracellular solution; 2) one cell in m guscn in water; or 3) ten cells in m guscn in water. the amount of rna in the cells was negligible compared to the externally added rna. the yield was unaffected by the extracellular solution and in fact increased in the presence of cells in guscn, a phenomenon investigated further. as an additional control for inhibitors, we added an equal amount of in vitro transcribed artificial rna, based on the cyclophilin e gene, to the lysis solution. ppie was measured in every single-cell sample and deviation >1% led to exclusion from further analysis. in figure 2b, an equal amount of purified rna was reverse transcribed in the presence of and mm guscn, followed by qpcr using five different primer pairs. the rt-reaction efficiency was significantly improved for all tested genes using mm guscn. by contrast, mm guscn had no effect whereas mm guscn was strongly inhibitory. we also evaluated the addition of – % 2-mercapto ethanol, but no effect on the rt yield, either alone or in concert with guscn, was observed. formation of correct pcr-products was confirmed by agarose gel electrophoresis. neither of the other two lysis conditions, np- or proteinase k, had any discernible effect on rt yield. taken together, we chose to use ~ m guscn in the single-cell lysis solution due to superior cell lysis ability and positive effect on the rt reaction at the concentration of mm. rt- and qpcr-induced noise the total technical noise level of rt-qpcr was estimated by diluting purified total rna from four to six different concentrations, ~10- copies of ribosomal protein s, ~50- copies of sry-box containing gene, nestin and glial fibrillary acidic protein and ~500-5· copies of ins transcripts). for each dilution, identical rt-qpcr reactions were run. the mrna concentrations were chosen to primarily cover low-medium abundance transcripts, i.e. chgb, gfap, nes, rps and sox consequently, the concentration of the high abundance ins transcript is 50– times higher. the qpcr-specific noise was calculated from qpcr replicates at five different concentrations of mouse cdna. the measurements were combined to create a model of rt- and qpcr-induced noise, as well as errors introduced by the liquid handling. the technical reproducibility of rt and qpcr is represented here by the noise strength, for different initial copy numbers. assuming that the noise from each reaction is additive, we can write the total noise as η2total = η2dilution + η2rt+ η2pcr and by fitting this model to the data, we can quantify the contribution of each component. η2dilution corresponds to poisson-type noise which arises when a sample with a low number of molecules is imperfectly diluted. additional noise is introduced by the rt-reaction and the final term corresponds to the noise introduced by the qpcr step. in additional file we derive functional forms for each component and we show how they can be assessed from replicate measurements. single-cell rt-qpcr using a glass capillary mounted on a micromanipulator, we collected pancreatic β-cells incubated in and mm glucose and emptied the intact cell in μl m guscn. following reverse transcription, we subjected each single-cell's cdna to qpcr using primers for rps chgb and ins. the median expression level across the different glucose concentrations for these three genes were and mrna copies per cell respectively. some cells failed to generate a signal. transcript level heterogeneity is high and the distribution is skewed, which is in agreement with previous studies of eukaryotic cells. one consequence of this heterogeneity is that the top 10% of the cells with the highest ins expression account for 50% of the total ins mrna. increasing glucose concentrations altered the median ins expression from mrna copies at mm glucose to and mrna copies for and mm glucose, respectively. the increase was mainly due to an increasing fraction of cells with very high expression levels. glucose did not have an effect on rps or chgb. how much of this variability stems from true, biological variation and how much is attributable to the rt-qpcr? the rt-qpcr noise model applied on the single-cell data suggests that the rt-qpcr noise is ~ % of the total variation in the ins expression. for rps which has lower expression, the range is 10–50% and for chgb 75–200%. thus, the decreasing accuracy of the rt-qpcr at lower copy numbers heavily influences measurements of low-abundance transcripts such as chgb. moreover, the 95% confidence level shows that a single measurement is accurate to within a factor of two when the expression level is > transcripts. we conclude that in the case of abundantly expressed genes, the distribution of the measurement errors is very narrow compared to the biological noise and the fluctuations introduced by the experimental procedure are negligible. finally, we investigated pcr inhibition caused by overloading the reaction with cdna. we tried to mitigate the pcr inhibition by decreasing the ratio between reverse transcriptase and taq polymerase, and found that a ratio ≤ was required to completely remove inhibitory effects. at low concentrations of rna, decreasing the rt-enzyme concentration did not have a negative effect on rt-yield. discussion individual cells have transcript levels which vary significantly from the population mean. the biological significance of this heterogeneity is still not clearly understood and it is an active area of research today. to characterize this cell-to-cell variation, high precision measurements of single cells are required. technically, such measurements are still very challenging and we have presented an rt-qpcr protocol which should be accessible for most laboratories. by using a purification-free protocol we are both simplifying single-cell measurements and eliminating rna losses associated with current available purification protocols. high concentrations of guanidine salts are commonly used in standard rna purification protocols, where it denatures nucleases rapidly and liberates nucleic acids from bound proteins. since guscn strongly inhibits downstream reactions, the rna is normally purified. at lower concentrations of guscn the effect diminishes, but rnases are completely inhibited even at m guscn and we show that the cell lysis ability remains strong at m. by ensuring sufficient dilution of the guscn prior reverse transcription, the concentration is brought down to regions where it does not interfere with the enzymatic reactions. indeed, we demonstrate that low concentrations even had a favourable effect on the rt yield and reproducibility. this effect may be a result from reduced secondary structures of the mrna, enhanced enzyme activity, and/or that guscn protects the mrna from degradation. we have also developed a mathematical model which allows us to quantify the contributions from the rt and pcr steps to the technical noise. the only parameter in our model which is not directly obtained from the experimental data is the rt efficiency. the rt efficiency can be estimated experimentally by rt-qpcr using known amounts of synthetic rna mimicking the properties of the native mrna. however, producing full length mrnas is challenging and it is common to use a truncated rna molecule which may not be reverse transcribed with the same efficiency as the native mrna. instead, we use our model to determine the rt efficiency indirectly; using non-linear regression we find the rt efficiency which best fits the data. we find that this indirect method gives realistic results for chgb, rps gfap, nes and sox and we believe that it could be a useful way of estimating the rt efficiency for a given gene. given the limited amount of mrna in a single cell, how should the rt and qpcr experiments be designed in order to minimize technical noise? we find that the benefit of replicate rt reactions is insignificant, depending on the rt efficiency, and we recommend a single rt reaction using all available mrna. in figure we illustrate different strategies to distribute the cdna in qpcr reactions. high dilution allows more genes and replicates to be analyzed, but this is at the price of lower mrna copy numbers, thereby increasing the measurement noise. figures 6a–b shows how the technical noise increases when the sample is diluted. curves show technical noise levels for each sample when it is split in or equal parts between rt and pcr. figures 6c–d show that additional dilution of the cdna to allow qpcr replicates is not a suitable approach to decrease the technical noise. in particular, for ins, which has a low rt efficiency there is a slight increase of the technical noise when the number of replicates is increased. in contrast, for rps with a high rt efficiency there is a slight reduction of the technical noise for low mrna numbers. in summary, the single-cell mrna should be reverse transcribed in a single rt reaction followed by one qpcr reaction per gene. it is advantageous to use more of the single-cell cdna for low-abundance genes and a smaller part for abundantly expressed genes. these conclusions are only valid for low mrna levels. however, loading large volumes of cdna to the qpcr may cause inhibition. in an effort to reduce this effect, we confirm previous findings suggesting a lowered reverse transcriptase concentration and an increased taq enzyme concentration. an alternate solution is to precipitate the single-cell cdna in ethanol, thus utilizing a larger fraction of the total amount in the qpcr. yet another option is multiplex pre-amplification rt-pcr for 10– cycles, followed by singleplex qpcr on the resulting pool of pcr-products. this could decrease technical noise levels, provided that the multiplex pcr is efficient, reproducible and unbiased. our model of the technical noise suggests that most of the noise in the method stems from the qpcr, at least when the initial template copy number is low and the rt-efficiency is relatively high. however, our analyses show that the rt-efficiencies vary significantly across genes and for low efficiencies, the rt reaction will constitute a larger proportion of the total noise. compared to the biological cell-to-cell variation, the technical noise is insignificant down to ~ copies, but becomes considerable at < rna copies. the model includes only three experimental factors; the rt-reaction, dilution of the sample and the qpcr while in reality there are many more steps contributing to the total noise. thus, our estimates of the noise components should be considered upper bounds since they will most likely become smaller if additional factors are included in the model.  we found that the strong denaturant guanidine thiocyanate can serve as lysis agent and rnase inhibitor and improve reverse transcription yield in purification-free single-cell rt-qpcr. our protocol allows fast and streamlined measurements of single-cell gene expression. analysis of technical noise caused by the rt- and pcr-reaction showed that this noise is insignificant compared to biological cell-to-cell variation at mrna copy numbers >~ below this level technical noise stemming from the pcr, which increases dramatically when initial copy number is <~ cdna copies, becomes significant. in addition, to achieve a high technical reproducibility, genes should be analyzed with lowest possible dilution between the rt and qpcr. that is, a single qpcr measurement should be performed for each gene, where the number of genes to be analyzed determines the total dilution factor between rt and pcr. 
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. importantly, the model allows us to determine the rt efficiency without using artificial rna as a standard. quantitative reverse transcription pcr is the most accessible method which provides sufficiently accurate measurements of mrna in single cells. the accuracy of the measurements was determined by a quantitative noise-model of the reverse transcription and pcr. 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. low concentration of guanidine thiocyanate was used to fully lyse single pancreatic β-cells followed by rt-qpcr without the need for purification. the noise is insignificant for initial copy numbers > 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.
 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 is the most accessible method which provides sufficiently accurate measurements of mrna in single cells.  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 > 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. 