ercc Search Results


gene exp ercc 00130 ac03459943 a1  (Thermo Fisher)
87
Thermo Fisher gene exp ercc 00130 ac03459943 a1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Gene Exp Ercc 00130 Ac03459943 A1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gene exp ercc 00130 ac03459943 a1/product/Thermo Fisher
Average 87 stars, based on 1 article reviews
gene exp ercc 00130 ac03459943 a1 - by Bioz Stars, 2026-04
87/100 stars
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gene exp ercc 00096 ac03460023 a1  (Thermo Fisher)
90
Thermo Fisher gene exp ercc 00096 ac03460023 a1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Gene Exp Ercc 00096 Ac03460023 A1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gene exp ercc 00096 ac03460023 a1/product/Thermo Fisher
Average 90 stars, based on 1 article reviews
gene exp ercc 00096 ac03460023 a1 - by Bioz Stars, 2026-04
90/100 stars
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gene exp ercc 00136 ac03459946 a1  (Thermo Fisher)
92
Thermo Fisher gene exp ercc 00136 ac03459946 a1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Gene Exp Ercc 00136 Ac03459946 A1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gene exp ercc 00136 ac03459946 a1/product/Thermo Fisher
Average 92 stars, based on 1 article reviews
gene exp ercc 00136 ac03459946 a1 - by Bioz Stars, 2026-04
92/100 stars
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gene exp ercc 00054 ac03459999 a1  (Thermo Fisher)
92
Thermo Fisher gene exp ercc 00054 ac03459999 a1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Gene Exp Ercc 00054 Ac03459999 A1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gene exp ercc 00054 ac03459999 a1/product/Thermo Fisher
Average 92 stars, based on 1 article reviews
gene exp ercc 00054 ac03459999 a1 - by Bioz Stars, 2026-04
92/100 stars
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gene exp ercc 00002 ac03459872 a1  (Thermo Fisher)
86
Thermo Fisher gene exp ercc 00002 ac03459872 a1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Gene Exp Ercc 00002 Ac03459872 A1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gene exp ercc 00002 ac03459872 a1/product/Thermo Fisher
Average 86 stars, based on 1 article reviews
gene exp ercc 00002 ac03459872 a1 - by Bioz Stars, 2026-04
86/100 stars
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gene exp ercc 00131 ac03459944 a1  (Thermo Fisher)
92
Thermo Fisher gene exp ercc 00131 ac03459944 a1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Gene Exp Ercc 00131 Ac03459944 A1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gene exp ercc 00131 ac03459944 a1/product/Thermo Fisher
Average 92 stars, based on 1 article reviews
gene exp ercc 00131 ac03459944 a1 - by Bioz Stars, 2026-04
92/100 stars
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gene exp ercc 00170 ac03460062 a1  (Thermo Fisher)
85
Thermo Fisher gene exp ercc 00170 ac03460062 a1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Gene Exp Ercc 00170 Ac03460062 A1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gene exp ercc 00170 ac03460062 a1/product/Thermo Fisher
Average 85 stars, based on 1 article reviews
gene exp ercc 00170 ac03460062 a1 - by Bioz Stars, 2026-04
85/100 stars
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gene exp ercc 00085 ac03459923 a1  (Thermo Fisher)
92
Thermo Fisher gene exp ercc 00085 ac03459923 a1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Gene Exp Ercc 00085 Ac03459923 A1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gene exp ercc 00085 ac03459923 a1/product/Thermo Fisher
Average 92 stars, based on 1 article reviews
gene exp ercc 00085 ac03459923 a1 - by Bioz Stars, 2026-04
92/100 stars
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gene exp ercc 00096 ac03459927 a1  (Thermo Fisher)
94
Thermo Fisher gene exp ercc 00096 ac03459927 a1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Gene Exp Ercc 00096 Ac03459927 A1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gene exp ercc 00096 ac03459927 a1/product/Thermo Fisher
Average 94 stars, based on 1 article reviews
gene exp ercc 00096 ac03459927 a1 - by Bioz Stars, 2026-04
94/100 stars
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gene exp ercc 00095 ac03459926 a1  (Thermo Fisher)
91
Thermo Fisher gene exp ercc 00095 ac03459926 a1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Gene Exp Ercc 00095 Ac03459926 A1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gene exp ercc 00095 ac03459926 a1/product/Thermo Fisher
Average 91 stars, based on 1 article reviews
gene exp ercc 00095 ac03459926 a1 - by Bioz Stars, 2026-04
91/100 stars
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antiercc1  (Boster Bio)
90
Boster Bio antiercc1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Antiercc1, supplied by Boster Bio, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/antiercc1/product/Boster Bio
Average 90 stars, based on 1 article reviews
antiercc1 - by Bioz Stars, 2026-04
90/100 stars
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gene exp ercc 00113 ac03459936 a1  (Thermo Fisher)
86
Thermo Fisher gene exp ercc 00113 ac03459936 a1
Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® <t>ERCC</t> RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).
Gene Exp Ercc 00113 Ac03459936 A1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gene exp ercc 00113 ac03459936 a1/product/Thermo Fisher
Average 86 stars, based on 1 article reviews
gene exp ercc 00113 ac03459936 a1 - by Bioz Stars, 2026-04
86/100 stars
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Image Search Results


Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® ERCC RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).

Journal: Regenerative Therapy

Article Title: β2-Microglobulin is an appropriate reference gene for RT-PCR-based gene expression analysis of hematopoietic stem cells

doi: 10.1016/j.reth.2015.04.003

Figure Lengend Snippet: Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® ERCC RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).

Article Snippet: The following pre-made TaqMan ® MGB probes and primer sets were purchased from Life Technologies: beta-actin ( Actb ) (Mm00607939_s1, Mm01205647_g1), B2m (Mm00437762_m1, Mm00437764_m1), glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1), hypoxanthine phosphoribosyl transferase ( Hprt ) (Mm01318741_m1, Mm00446968_m1), ERCC-00130 (Ac03459943_a1), ERCC-00002 (Ac03459872_a1), ERCC-00113 (Ac03459936_a1).

Techniques: Gene Expression, Control, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, FACS

B2m is stably expressed in HSCs, even after ex vivo culture. (A) CD150 + CD34 − KSL cells were cultured for 5 days under 3 different conditions in the presence of SCF and/or TPO. After the culture, CD48 − KSL cells, referred to as the cultured HSC population , were clonally sorted and subsequently subjected to single-cell gene expression analysis. (B) Frequency of cells in which the expression of the indicated genes could be detected before and after culture. The numbers in parentheses represent cells that had positive signals/examined cells under each condition. (C) Heat maps showing the expression values of the indicated genes in individual HSCs before and after the culture under the 3 different conditions. The expression levels of these genes were normalized to the Ct values of ERCC-00130, an exogenously added control RNA. (Fresh: n = 159, SCF + TPO: n = 148, SCF: n = 163, TPO: n = 127).

Journal: Regenerative Therapy

Article Title: β2-Microglobulin is an appropriate reference gene for RT-PCR-based gene expression analysis of hematopoietic stem cells

doi: 10.1016/j.reth.2015.04.003

Figure Lengend Snippet: B2m is stably expressed in HSCs, even after ex vivo culture. (A) CD150 + CD34 − KSL cells were cultured for 5 days under 3 different conditions in the presence of SCF and/or TPO. After the culture, CD48 − KSL cells, referred to as the cultured HSC population , were clonally sorted and subsequently subjected to single-cell gene expression analysis. (B) Frequency of cells in which the expression of the indicated genes could be detected before and after culture. The numbers in parentheses represent cells that had positive signals/examined cells under each condition. (C) Heat maps showing the expression values of the indicated genes in individual HSCs before and after the culture under the 3 different conditions. The expression levels of these genes were normalized to the Ct values of ERCC-00130, an exogenously added control RNA. (Fresh: n = 159, SCF + TPO: n = 148, SCF: n = 163, TPO: n = 127).

Article Snippet: The following pre-made TaqMan ® MGB probes and primer sets were purchased from Life Technologies: beta-actin ( Actb ) (Mm00607939_s1, Mm01205647_g1), B2m (Mm00437762_m1, Mm00437764_m1), glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1), hypoxanthine phosphoribosyl transferase ( Hprt ) (Mm01318741_m1, Mm00446968_m1), ERCC-00130 (Ac03459943_a1), ERCC-00002 (Ac03459872_a1), ERCC-00113 (Ac03459936_a1).

Techniques: Stable Transfection, Ex Vivo, Cell Culture, Gene Expression, Expressing, Control

The expression level of B2m in HSCs is relatively independent of the influence of ex vivo culture. Using the results of single-cell real-time RT-PCR, changes in the expression levels of the indicated genes in HSCs were examined after ex vivo culture. The expression levels of these genes in individual single-cell samples were normalized to the Ct values of ERCC-00130, an external control RNA. The graphs show relative expression values of Actb (A), B2m (B), Gapdh (C) and Hprt (D). Data are shown as mean ± S.D. (** p < 0.05, Fresh: n = 159, SCF + TPO: n = 148, SCF: n = 163, TPO: n = 127).

Journal: Regenerative Therapy

Article Title: β2-Microglobulin is an appropriate reference gene for RT-PCR-based gene expression analysis of hematopoietic stem cells

doi: 10.1016/j.reth.2015.04.003

Figure Lengend Snippet: The expression level of B2m in HSCs is relatively independent of the influence of ex vivo culture. Using the results of single-cell real-time RT-PCR, changes in the expression levels of the indicated genes in HSCs were examined after ex vivo culture. The expression levels of these genes in individual single-cell samples were normalized to the Ct values of ERCC-00130, an external control RNA. The graphs show relative expression values of Actb (A), B2m (B), Gapdh (C) and Hprt (D). Data are shown as mean ± S.D. (** p < 0.05, Fresh: n = 159, SCF + TPO: n = 148, SCF: n = 163, TPO: n = 127).

Article Snippet: The following pre-made TaqMan ® MGB probes and primer sets were purchased from Life Technologies: beta-actin ( Actb ) (Mm00607939_s1, Mm01205647_g1), B2m (Mm00437762_m1, Mm00437764_m1), glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1), hypoxanthine phosphoribosyl transferase ( Hprt ) (Mm01318741_m1, Mm00446968_m1), ERCC-00130 (Ac03459943_a1), ERCC-00002 (Ac03459872_a1), ERCC-00113 (Ac03459936_a1).

Techniques: Expressing, Ex Vivo, Quantitative RT-PCR, Control

Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® ERCC RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).

Journal: Regenerative Therapy

Article Title: β2-Microglobulin is an appropriate reference gene for RT-PCR-based gene expression analysis of hematopoietic stem cells

doi: 10.1016/j.reth.2015.04.003

Figure Lengend Snippet: Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® ERCC RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).

Article Snippet: The following pre-made TaqMan ® MGB probes and primer sets were purchased from Life Technologies: beta-actin ( Actb ) (Mm00607939_s1, Mm01205647_g1), B2m (Mm00437762_m1, Mm00437764_m1), glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1), hypoxanthine phosphoribosyl transferase ( Hprt ) (Mm01318741_m1, Mm00446968_m1), ERCC-00130 (Ac03459943_a1), ERCC-00002 (Ac03459872_a1), ERCC-00113 (Ac03459936_a1).

Techniques: Gene Expression, Control, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, FACS

B2m is stably expressed in HSCs, even after ex vivo culture. (A) CD150 + CD34 − KSL cells were cultured for 5 days under 3 different conditions in the presence of SCF and/or TPO. After the culture, CD48 − KSL cells, referred to as the cultured HSC population , were clonally sorted and subsequently subjected to single-cell gene expression analysis. (B) Frequency of cells in which the expression of the indicated genes could be detected before and after culture. The numbers in parentheses represent cells that had positive signals/examined cells under each condition. (C) Heat maps showing the expression values of the indicated genes in individual HSCs before and after the culture under the 3 different conditions. The expression levels of these genes were normalized to the Ct values of ERCC-00130, an exogenously added control RNA. (Fresh: n = 159, SCF + TPO: n = 148, SCF: n = 163, TPO: n = 127).

Journal: Regenerative Therapy

Article Title: β2-Microglobulin is an appropriate reference gene for RT-PCR-based gene expression analysis of hematopoietic stem cells

doi: 10.1016/j.reth.2015.04.003

Figure Lengend Snippet: B2m is stably expressed in HSCs, even after ex vivo culture. (A) CD150 + CD34 − KSL cells were cultured for 5 days under 3 different conditions in the presence of SCF and/or TPO. After the culture, CD48 − KSL cells, referred to as the cultured HSC population , were clonally sorted and subsequently subjected to single-cell gene expression analysis. (B) Frequency of cells in which the expression of the indicated genes could be detected before and after culture. The numbers in parentheses represent cells that had positive signals/examined cells under each condition. (C) Heat maps showing the expression values of the indicated genes in individual HSCs before and after the culture under the 3 different conditions. The expression levels of these genes were normalized to the Ct values of ERCC-00130, an exogenously added control RNA. (Fresh: n = 159, SCF + TPO: n = 148, SCF: n = 163, TPO: n = 127).

Article Snippet: The following pre-made TaqMan ® MGB probes and primer sets were purchased from Life Technologies: beta-actin ( Actb ) (Mm00607939_s1, Mm01205647_g1), B2m (Mm00437762_m1, Mm00437764_m1), glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1), hypoxanthine phosphoribosyl transferase ( Hprt ) (Mm01318741_m1, Mm00446968_m1), ERCC-00130 (Ac03459943_a1), ERCC-00002 (Ac03459872_a1), ERCC-00113 (Ac03459936_a1).

Techniques: Stable Transfection, Ex Vivo, Cell Culture, Gene Expression, Expressing, Control

The expression level of B2m in HSCs is relatively independent of the influence of ex vivo culture. Using the results of single-cell real-time RT-PCR, changes in the expression levels of the indicated genes in HSCs were examined after ex vivo culture. The expression levels of these genes in individual single-cell samples were normalized to the Ct values of ERCC-00130, an external control RNA. The graphs show relative expression values of Actb (A), B2m (B), Gapdh (C) and Hprt (D). Data are shown as mean ± S.D. (** p < 0.05, Fresh: n = 159, SCF + TPO: n = 148, SCF: n = 163, TPO: n = 127).

Journal: Regenerative Therapy

Article Title: β2-Microglobulin is an appropriate reference gene for RT-PCR-based gene expression analysis of hematopoietic stem cells

doi: 10.1016/j.reth.2015.04.003

Figure Lengend Snippet: The expression level of B2m in HSCs is relatively independent of the influence of ex vivo culture. Using the results of single-cell real-time RT-PCR, changes in the expression levels of the indicated genes in HSCs were examined after ex vivo culture. The expression levels of these genes in individual single-cell samples were normalized to the Ct values of ERCC-00130, an external control RNA. The graphs show relative expression values of Actb (A), B2m (B), Gapdh (C) and Hprt (D). Data are shown as mean ± S.D. (** p < 0.05, Fresh: n = 159, SCF + TPO: n = 148, SCF: n = 163, TPO: n = 127).

Article Snippet: The following pre-made TaqMan ® MGB probes and primer sets were purchased from Life Technologies: beta-actin ( Actb ) (Mm00607939_s1, Mm01205647_g1), B2m (Mm00437762_m1, Mm00437764_m1), glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1), hypoxanthine phosphoribosyl transferase ( Hprt ) (Mm01318741_m1, Mm00446968_m1), ERCC-00130 (Ac03459943_a1), ERCC-00002 (Ac03459872_a1), ERCC-00113 (Ac03459936_a1).

Techniques: Expressing, Ex Vivo, Quantitative RT-PCR, Control