The quantity of the vimentin gene expression (V2258) in the adult testicular cells was assayed by Fluidigm (dynamic array chips). Glyceraldehyde-3-phosphate dehydrogenase was utilized as a housekeeping gene for standardization. Cultured testicular cells were selected with a micromanipulator, and lysed with a lysis buffer solution containing 1.3 μL TE buffer, 9 μL RT-PreAmp Master Mix, 0.2 μL R.T./Taq Superscript III (Invitrogen, Waltham, MA, USA), 2.5 μL 0.2× assay pool, and 5.0 μL Cells Direct 2× Reaction Mix (Invitrogen, Waltham, MA, USA). The targeted transcripts were quantified using TaqMan real-time PCR on the Biomark real-time quantitative PCR system (Fluidigm-PCR). Cells were examined in two technical repeats. Ct evaluation was analyzed by GenEx software (version 5.4.2, MultiD Analyses, Gothenburg, Sweden) [3 (link),16 (link)].
Rt preamp master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
RT-PreAmp Master Mix is a pre-formulated reagent mix designed for use in reverse transcription and pre-amplification steps prior to real-time PCR or digital PCR analysis. The mix contains all the necessary components, including reverse transcriptase and a DNA polymerase, to efficiently convert RNA to cDNA and perform limited-cycle pre-amplification.
Automatically generated - may contain errors
Lab products found in correlation
Cells direct 2 reaction mix, by Thermo Fisher Scientific (8 mentions)
Rt taq superscript 3, by Thermo Fisher Scientific (7 mentions)
Biomark system, by Standard BioTools (4 mentions)
Taqman assay, by Thermo Fisher Scientific (3 mentions)
Dynamic array chips, by Standard BioTools (2 mentions)
Biomark real time pcr analysis software, by Standard BioTools (1 mentions)
Biomark data collection software, by Standard BioTools (1 mentions)
Superscript 3 rt platinum taq mix, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
11 protocols using rt preamp master mix
1
Quantifying Vimentin Gene Expression in Adult Testicular Cells
2
Quantifying PLZF Expression in Stem Cells
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Quantity of the PLZF gene expression (Mm01176868_
m1) in the neonate SSCs, adult SSCs, ES cells, and
ES-like cells were examined by dynamic array chips(Fluidigm). Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH, Mm99999915_g1) was used as housekeepinggene for normalization. Cultured cells were selected with amicromanipulator, lysed with lysis buffer solution containing
1.3 µl TE buffer, 0.2 µl RT/Taq Superscript III (Invitrogen,
USA), 9 µl RT-PreAmp Master Mix, 5.0 µl Cells Direct2× Reaction Mix (Invitrogen, USA), and 2.5 µl 0.2× assay
pool. Using TaqMan real-time PCR on the BioMark Real-
Time quantitative PCR (qPCR) system, the amount of RNA-
targeted copies was evaluated. Samples were examined intwo technical repeats. The Ct values were analyzed by GenEx
software from the MultiD analysis (2 (link), 3 (link), 6 (link)).
m1) in the neonate SSCs, adult SSCs, ES cells, and
ES-like cells were examined by dynamic array chips(Fluidigm). Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH, Mm99999915_g1) was used as housekeepinggene for normalization. Cultured cells were selected with amicromanipulator, lysed with lysis buffer solution containing
1.3 µl TE buffer, 0.2 µl RT/Taq Superscript III (Invitrogen,
USA), 9 µl RT-PreAmp Master Mix, 5.0 µl Cells Direct2× Reaction Mix (Invitrogen, USA), and 2.5 µl 0.2× assay
pool. Using TaqMan real-time PCR on the BioMark Real-
Time quantitative PCR (qPCR) system, the amount of RNA-
targeted copies was evaluated. Samples were examined intwo technical repeats. The Ct values were analyzed by GenEx
software from the MultiD analysis (2 (link), 3 (link), 6 (link)).
3
Single-cell mRNA profiling of neural cells
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A TaqMan assay pool was prepared by adding each of the 48 TaqMan assays (20×; Applied Biosystems) to a final concentration of 0.2× for each assay. Neurospheres were dissociated and single cells were sorted by fluorescence-activated cell sorting (FACS) directly into 10 μL of RT-PreAmp Master Mix (5 μL CellsDirect 2× Reaction Mix [Invitrogen], 2.5 μL 0.2× Assay pool, 0.5 μL SuperScript® III RT/Platinum® Taq mix [Invitrogen], and 2 μL TE buffer). Cells were frozen at −80°C and thawed to induce lysis. Sequence-specific reverse transcription (50°C for 20 min) and reverse transcriptase inactivation (95°C for 2 min) were performed to generate complementary DNAs (cDNAs) of the 48 genes, following which sequence-specific preamplification (18 cycles at 95°C for 15 s and 60°C for 4 min) was performed. The preamplified cDNA was diluted 5-fold and used for single-cell mRNA profiling in 48.48 dynamic arrays on a BioMark system (Fluidigm). Single-cell mRNA profiling was run using the BioMark Data Collection software (Fluidigm) and Ct values were calculated using the BioMark Real-time PCR analysis software (Fluidigm). Cells with Ct value for the endogenous control β-actin between 15 and 25 were considered for analysis. Ct values for a specific cell were normalized to the endogenous control by subtracting the Ct value of β-actin for the same cell. The assumed baseline Ct value is 31.
4
Expression Analysis of DDX4 in SSCs
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Measurements of the expression of the gene DEAD
(Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4 or VASA)
Mm00802445_m1 in the neonate and adult SSCs were analyzed
with Dynamic Array chips (Fluidigm). A housekeeping
gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Mm99999915_g1 was used for normalization, in different types
of cultured cells. SSCs were picked with a micromanipulator,
lysed with a lysis buffer solution containing 9 μl RT-PreAmp
Master Mix (5.0 μl Cells Direct 2× Reaction Mix) (Invitrogen,
USA), 2.5 μl 0.2× assay pool, 0.2 μl RT/Taq Superscript III
(Invitrogen, USA) and 1.3 μl TE buffer. The number of RNAtargeted
transcripts was measured using TaqMan PCR assays
on the BioMark Real-Time quantitative PCR (qPCR) system.
Each sample was analyzed in two technical repeats. The Ct
values were examined using GenEx software from MultiD for
analysis (4 (link), 5 (link)).
(Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4 or VASA)
Mm00802445_m1 in the neonate and adult SSCs were analyzed
with Dynamic Array chips (Fluidigm). A housekeeping
gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Mm99999915_g1 was used for normalization, in different types
of cultured cells. SSCs were picked with a micromanipulator,
lysed with a lysis buffer solution containing 9 μl RT-PreAmp
Master Mix (5.0 μl Cells Direct 2× Reaction Mix) (Invitrogen,
USA), 2.5 μl 0.2× assay pool, 0.2 μl RT/Taq Superscript III
(Invitrogen, USA) and 1.3 μl TE buffer. The number of RNAtargeted
transcripts was measured using TaqMan PCR assays
on the BioMark Real-Time quantitative PCR (qPCR) system.
Each sample was analyzed in two technical repeats. The Ct
values were examined using GenEx software from MultiD for
analysis (4 (link), 5 (link)).
5
Profiling Gene Expression in SSCs and TSCs
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The expression levels of the DAZL (Mm00515630_m1), VASA (Mm00802445_m1), TAF4B (Mm01254136_m1) and Zbtb16 (Mm01176868_m1) genes in the SSCs and TSC cells were examined by the Fluidigm biomark system. SSCs and TSCs were picked up with a micromanipulator technique and lysed with a lysis buffer solution containing 9 μl of RT-PreAmp Master Mix (5.0 μl of Cells Direct 2 × Reaction Mix, Invitrogen, USA), 2.5 μl of 0.2 × assay pool, 1.3 μl of TE buffer, and 0.2 μl of RT/Taq Superscript III (Invitrogen, USA). The amounts of amplified product of RNA-targeted copies were then examined with TaqMan RT-PCR on the BioMark Fluidigm RT-PCR system. Samples were analyzed in two technical repeats. The Ct values were calculated using the Excel and GenEx software.
6
Pluripotency Gene Expression Analysis
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The expression of various pluripotency- and germ cellassociated
genes Oct4, Nanog, Sox2, Klf4, c-Myc, Lin28,
Gdf3, Tdgf1, Dppa-5, Stra8 and Gpr-125 was analyzed
utilizing dynamic array chips (Table 1 ). The housekeeping
gene, Gapdh, was selected for normalization of data in
different cultured cell types, including ESCs, ES-like
cells, epiblast-like cells and transitional colonies. The
expression fold change of mRNA was compared to mouse
embryonic fibroblasts (MEF) feeder cells as an additional
control. With the help of a micromanipulator (Narashige
Instruments) about 50 cells were manually selected from
each sample. Afterwards, the selected cells were lysed with
a special lysis buffer containing 9 μl RT-PreAmp Master
Mix (5.0 μl Cells Direct 2× Reaction Mix) (Invitrogen,
USA), 2.5 μl 0.2× assay pool, 0.2 μl RT/Taq Superscript
III (Invitrogen, USA) and 1.3 μl TE buffer and directly
frozen and stored at -80˚C. The targeted transcripts were
quantified with TaqMan real-time PCR on the BioMark
real-time quantitative PCR (qPCR) system (Fluidigm,
USA), with TaqMan gene expression assays (Invitrogen,
USA) in 48.48 dynamic arrays. Two technical replicates
were processed to analyze every sample. The CT values
were analysed with GenEx software from MultiD, Excel
and SPSS (1 (link), 3 (link), 11 ).
genes Oct4, Nanog, Sox2, Klf4, c-Myc, Lin28,
Gdf3, Tdgf1, Dppa-5, Stra8 and Gpr-125 was analyzed
utilizing dynamic array chips (
gene, Gapdh, was selected for normalization of data in
different cultured cell types, including ESCs, ES-like
cells, epiblast-like cells and transitional colonies. The
expression fold change of mRNA was compared to mouse
embryonic fibroblasts (MEF) feeder cells as an additional
control. With the help of a micromanipulator (Narashige
Instruments) about 50 cells were manually selected from
each sample. Afterwards, the selected cells were lysed with
a special lysis buffer containing 9 μl RT-PreAmp Master
Mix (5.0 μl Cells Direct 2× Reaction Mix) (Invitrogen,
USA), 2.5 μl 0.2× assay pool, 0.2 μl RT/Taq Superscript
III (Invitrogen, USA) and 1.3 μl TE buffer and directly
frozen and stored at -80˚C. The targeted transcripts were
quantified with TaqMan real-time PCR on the BioMark
real-time quantitative PCR (qPCR) system (Fluidigm,
USA), with TaqMan gene expression assays (Invitrogen,
USA) in 48.48 dynamic arrays. Two technical replicates
were processed to analyze every sample. The CT values
were analysed with GenEx software from MultiD, Excel
and SPSS (1 (link), 3 (link), 11 ).
7
Quantifying GFRa1 Expression in Stem Cells
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The expression level of the GFRa1 Mm01253716_m1 gene in SSCs and TSCs
was examined by the Fluidigm Biomark system. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) Mm99999915-g1 was the reference gene for normalization. SSCs and TSCs were picked
up with a micromanipulator technique, lysed with a solution of lysis buffer that contained
9 μl RT-PreAmp Master Mix (5.0 μl Cells Direct 2× Reaction Mix, Invitrogen, USA), 2.5 μl
0.2× assay pool and 1.3 μl TE buffer, 0.2 μl RT/Taq Superscript III (Invitrogen, USA).
Then, the amount of the amplified product of RNA-targeted copies was examined with TaqMan
real-time PCR on a BioMark Real-Time Quantitative PCR (qPCR) system. Samples were analysed
in two technical repeats. The Ct values were calculated using Excel and GenEx software
(20 -22 (link)).
was examined by the Fluidigm Biomark system. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) Mm99999915-g1 was the reference gene for normalization. SSCs and TSCs were picked
up with a micromanipulator technique, lysed with a solution of lysis buffer that contained
9 μl RT-PreAmp Master Mix (5.0 μl Cells Direct 2× Reaction Mix, Invitrogen, USA), 2.5 μl
0.2× assay pool and 1.3 μl TE buffer, 0.2 μl RT/Taq Superscript III (Invitrogen, USA).
Then, the amount of the amplified product of RNA-targeted copies was examined with TaqMan
real-time PCR on a BioMark Real-Time Quantitative PCR (qPCR) system. Samples were analysed
in two technical repeats. The Ct values were calculated using Excel and GenEx software
(20 -22 (link)).
8
Quantifying Oct4 Expression in Stem Cells
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Quantity of the Oct4 gene expression (Mm03053917_g1) in SSCs, ES-like, EBs, Sertoli cells, and Mouse Embryonic Fibroblasts (MEF, for control) was assayed by Fluidigm dynamic array chips. Glyceraldehyde-3-phosphate dehydrogenase (Mm99999915_g1) was utilized as a housekeeping gene for normalization. All cells were selected with a micromanipulator, lysed with a lysis buffer solution containing 1.3 μl TE buffer, 9 μl RT-PreAmp Master Mix, 0.2 μl R.T./Taq Superscript III (Invitrogen, USA), 2.5 μl 0.2× assay pool, and 5.0 μl Cells Direct 2× Reaction Mix (Invitrogen, USA). The targeted transcripts were quanti ed using TaqMan real-time PCR on the Biomark Real-Time quantitative PCR system (Fluidigm-PCR) (
9
Quantitative PCR of Single-Cell RNA
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PMA and ionomycin in vitro-stimulated CD4+ CD25high T cells were single-cell sorted directly into RT-PreAmp Master Mix (Life Technologies). Cell lysis, sequence-specific reverse-transcription and sequence-specific amplification of cDNA were done as previously described37 (link) and high-throughput quantitative PCR was done on 96.96 Dynamic Arrays with a BioMark system (Fluidigm San Francisco, CA, USA). Amplification curves were quality filtered-using a threshold of 0.65, and melting curves generated for each reaction were manually inspected as an added quality control step. Cycling threshold (CT) values were calculated with BioMark system software for each assay and the same thresholds were used across all experiments. After exclusion of failed reactions, cells in which the expression of at least three different housekeeping genes mRNA was detected were retained for further analysis. All primers (Fluidigm DELTA gene assays) were pre-validate using positive and negative single cells (data not shown). ΔCt values were calculated in reference to the housekeeping genes.
10
Single-cell gene expression profiling
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Inventoried TaqMan assays (Life Technologies) were pooled to a final concentration of 0.2X for each of the 94 gene expression assays. Single CD8+ T cells were sorted directly into RT-PreAmp Master Mix (Life Technologies) containing the pooled assays. Cell lysis, sequence-specific RT, and sequence-specific amplification of cDNA were performed as previously described14 (link), and analyzed in 96.96 Dynamic Arrays on a BioMark system (Fluidigm). Ct values were calculated from the BioMark system software. Cells in which both Actb and Rn18s mRNA expression were detected were retained for further analyses.
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