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 taq superscript 3
Manufactured by Thermo Fisher Scientific
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The RT/Taq Superscript III is a laboratory equipment product that combines reverse transcriptase and DNA polymerase enzymes for the purpose of performing reverse transcription and PCR amplification in a single reaction.
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7 protocols using rt taq superscript 3
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
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)).
4
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.
5
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 ).
6
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)).
7
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) (
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