For S. cerevisiae we outline a protocol for the visualization of
mRNAs labeled with the low-affinity 24xMBSV6 (see Subheadings
2.1 and 3.1). The constitutive and well-characterized mRNA
MDN1 is used as a model gene. MDN1 was endogenously tagged
with 24xMBSV6 as previously described in [58]. Here, we detail
how to transform yeast cells with the constructs expressing
MCP-FP (Subheading 3.1.1), grow cells for live imaging (Subheading 3.1.2), prepare the coated dishes (Subheading 3.1.3),
perform live imaging (Subheading 3.1.4), and count single
mRNAs in living cells (Subheading 3.1.5).
For mammalian cells, we outline a protocol for the visualization
of mRNAs labeled with the low-affinity MS2x64 reporter, which is
particularly useful to analyze transcription dynamics (see Subheadings 2.2 and 3.2). To this end, we used a stable HeLa H9 AAVS1Tat cell line expressing the HIV-1 MS2x64 reporter gene
[60]. Here, we describe how to produce lentiviruses encoding the
MCP-FP (Subheading 3.2.1), how to generate a stable cell line
expressing the MCP-FP construct (Subheading 3.2.2), how to
1
1,1
1,2
1,3
1,4
1,5
1,6
NLS-MCP-eGFP
NLS-MCP-mEGFP
NLS-MCP-GFPEnvy
NLS-MCP-muGFP
NLS-MCP-sfGFP
NLS-MCP-mNG
Signal-to-noise-ratio
NS
NS
Fig. 5 Signal-to-noise ratio (SNR) of single RNA molecule detection with MCP-FP
variants. The bar plots show the mean values of the SNR of single RNA
molecules (error bar: standard deviation). NS: nonsignificant detection of single
RNAs occurred with the NLS-MCP-muGFP and NLS-MCP-mNG constructs. To
measure the intensity of single RNA molecules, a straight line with a width of
2 pixels was used, and the maximal value of the intensity plot was recorded. This
value was divided over the mean gray value measured in the nucleus of the cell
following the application of a Gaussian blur of 3 pixels (maximum intensity
projected images). The background value (outside the cell nucleus) was subtracted from each of these values (total of 133 single molecules quantified per
construct)
Imaging Single mRNAs in Living Eukaryotic Cells
129
mRNAs labeled with the low-affinity 24xMBSV6 (see Subheadings
2.1 and 3.1). The constitutive and well-characterized mRNA
MDN1 is used as a model gene. MDN1 was endogenously tagged
with 24xMBSV6 as previously described in [58]. Here, we detail
how to transform yeast cells with the constructs expressing
MCP-FP (Subheading 3.1.1), grow cells for live imaging (Subheading 3.1.2), prepare the coated dishes (Subheading 3.1.3),
perform live imaging (Subheading 3.1.4), and count single
mRNAs in living cells (Subheading 3.1.5).
For mammalian cells, we outline a protocol for the visualization
of mRNAs labeled with the low-affinity MS2x64 reporter, which is
particularly useful to analyze transcription dynamics (see Subheadings 2.2 and 3.2). To this end, we used a stable HeLa H9 AAVS1Tat cell line expressing the HIV-1 MS2x64 reporter gene
[60]. Here, we describe how to produce lentiviruses encoding the
MCP-FP (Subheading 3.2.1), how to generate a stable cell line
expressing the MCP-FP construct (Subheading 3.2.2), how to
1
1,1
1,2
1,3
1,4
1,5
1,6
NLS-MCP-eGFP
NLS-MCP-mEGFP
NLS-MCP-GFPEnvy
NLS-MCP-muGFP
NLS-MCP-sfGFP
NLS-MCP-mNG
Signal-to-noise-ratio
NS
NS
Fig. 5 Signal-to-noise ratio (SNR) of single RNA molecule detection with MCP-FP
variants. The bar plots show the mean values of the SNR of single RNA
molecules (error bar: standard deviation). NS: nonsignificant detection of single
RNAs occurred with the NLS-MCP-muGFP and NLS-MCP-mNG constructs. To
measure the intensity of single RNA molecules, a straight line with a width of
2 pixels was used, and the maximal value of the intensity plot was recorded. This
value was divided over the mean gray value measured in the nucleus of the cell
following the application of a Gaussian blur of 3 pixels (maximum intensity
projected images). The background value (outside the cell nucleus) was subtracted from each of these values (total of 133 single molecules quantified per
construct)
Imaging Single mRNAs in Living Eukaryotic Cells
129