Advanced

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

Bengtsson, Martin LU ; Hemberg, Martin; Rorsman, Patrik and Stålberg, Anders LU (2008) In BMC Molecular Biology 9(63).
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
BMC Molecular Biology
volume
9
issue
63
publisher
BioMed Central
external identifiers
  • wos:000258346800001
  • scopus:48249156189
ISSN
1471-2199
DOI
10.1186/1471-2199-9-63
language
English
LU publication?
yes
id
68dd91fa-6b8b-404a-a7ba-d029767f9125 (old id 1252906)
date added to LUP
2008-11-03 13:09:58
date last changed
2017-08-27 04:51:28
@article{68dd91fa-6b8b-404a-a7ba-d029767f9125,
  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.},
  author       = {Bengtsson, Martin and Hemberg, Martin and Rorsman, Patrik and Stålberg, Anders},
  issn         = {1471-2199},
  language     = {eng},
  number       = {63},
  publisher    = {BioMed Central},
  series       = {BMC Molecular Biology},
  title        = {Quantification of mRNA in single cells and modelling of RT-qPCR induced noise},
  url          = {http://dx.doi.org/10.1186/1471-2199-9-63},
  volume       = {9},
  year         = {2008},
}