The uteri were briefly rinsed with warmed Hank’s Balanced Salt Solution (HBSS, Invitrogen, Carlsbad, CA, USA) and the embryos were flushed by irrigation of flushing medium (Origio, Målov, Denmark) into uterine horns. Four to ten embryos were obtained from each mouse and three healthy blastocysts (dpc 3.0-4.0) were selected per mouse, pooled and cultured in BlastGen™ (Origio, Målov, Denmark). After 24 h of culture, blastocysts were treated with 10−7 M P4 or MPA for another 24 h. The embryos were assigned to each group (control, P4 or MPA-treated). Each embryo was mixed with single cell-to-CT™ qRT-PCR kit (Ambion, Waltham, MA, USA) buffer and denatured for qRT-PCR. For immunostaining, embryos were fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature and used for further processing.
Single cell to ct qrt pcr kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Single Cell-to-Ct qRT-PCR kit is a tool designed for the analysis of gene expression in individual cells. It enables the direct quantification of target gene expression from single cells using real-time PCR (qRT-PCR) technology.
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Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Sso universal sybr green supermix, by Bio-Rad (2 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
C1 single cell auto prep ifc for preamp, by Standard BioTools (1 mentions)
Biomark hd system, by Standard BioTools (1 mentions)
Real time pcr analysis software, by Standard BioTools (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Luna universal one step rt qpcr kit, by New England Biolabs (1 mentions)
Acid tyrode s solution, by Merck Group (1 mentions)
Sybr fast qpcr master mix, by Roche (1 mentions)
Iscript advanced reverse transcriptase, by Bio-Rad (1 mentions)
Abi prism 7500, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Sequence detection system software, by Bio-Rad (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Cfx96 touch real time pcr system, by Bio-Rad (1 mentions)
14 protocols using single cell to ct qrt pcr kit
1
Blastocyst Culture and Treatment with Progestins
2
Single-cell qPCR analysis of endolymphatic sac cells
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For single-cell qPCR, we captured single cells of P5 endolymphatic sacs using medium-sized microfluidic chips (C1 Single-Cell Auto Prep IFC for PreAmp) as outlined in the Fluidigm protocol (PN 100–6117 G1). Mixes for lysis, RT, and specific target amplification were prepared from the Single Cell-to-Ct qRT-PCR kit (Ambion, Austin, TX) and pre-designed TaqMan gene expression assays (Thermo Fisher Sientific). In addition to canonical cell markers, putative MRC and RRC markers at P5 (Figure 2—source data 3 ) were selected for analysis in an arbitrary manner.
After 18 cycles of preamplification, expression levels were measured by qPCR on a 96.96 Dynamic Array IFC using the Fluidigm BioMark HD system. cDNA from single cells was selected for qPCR in the same way as it was selected for RNA-seq. The threshold of cycles (Ct) values were calculated with Fluidigm Real-time PCR analysis software (RRID:SCR_15686 ) with the following settings: quality threshold of 0.65; a linear (derivative) baseline correction; and auto (detectors) method. We defined gene expression levels as log2 expression = LOD – Ct, in which we set Ct = 28 as LOD. We used the log2 expression dataset for hierarchical clustering.
After 18 cycles of preamplification, expression levels were measured by qPCR on a 96.96 Dynamic Array IFC using the Fluidigm BioMark HD system. cDNA from single cells was selected for qPCR in the same way as it was selected for RNA-seq. The threshold of cycles (Ct) values were calculated with Fluidigm Real-time PCR analysis software (RRID:
3
Single-cell RT-qPCR analysis of intestinal CD4+ T cells
4
Quantifying T Cell Gene Expression
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Transduced primary T cells were sorted by fluorescence-activated cell sorting (FACS) for retroviral reporter expression (viable and GFP positive population) 72 h after transduction. Sorted cells were directly processed using the Single Cell-to-CT™ qRT-PCR Kit (Ambion, 4458236) according to the manufacturer’s guidelines. qPCR analysis was performed by using the Luna® Universal One-Step RT-qPCR Kit (NEB, E3005S) according to the manufacturer’s instructions. Rpl19 gene was used as the loading control. All reactions were done in triplicates. Primer sequences were listed: mIl-2 F: 5-ATCAGCAATATCAGAGTAACTGTTGTA-3′; mIl-2 R: 5′-CATCTCCTCAGAAAGTCCACC-3′; mInpp5e F: 5′-GATCTTTCAGCCTTCTGGCCC-3′; mInpp5e R: 5′-GAGAGCCATGTTTCGGTCTG-3′; mRpl19 F: 5′-GAAATCGCCAATGCCAACTC-3′; mRpl19 R: 5′- TCCTTGGTCTTAGACCTGCG-3′.
5
Single-cell RNA-seq of Mouse Embryos
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The zona pellucida was removed by treatment with acid Tyrode’s solution (Sigma) and single cells from each preimplantation stage were collected using a micromanipulator. Total RNA isolation and cDNA synthesis were performed using a Single Cell-to-CT™ qRT-PCR Kit (Thermo Fisher) with slight modifications. In brief, half volumes of all reagents were used in this study. The qPCR analysis using TaqMan probes (Rnf12: Mm00488044_m1 Xist: Mm01232884_m1) was conducted without a cDNA preamplification step. A total of 4 or more embryos were randomly selected from which to collect single cells used in the assay. The remaining cells at the 2-cell stage in fertilized embryos were subjected to DNA-FISH analysis as described below.
6
Single-Cell Quantification of Glutamate Receptor
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Individual patched cells were processed following the provider´s recommendation for the Single Cell-to-CT qRT-PCR Kit (ThermoFisher Scientific). cDNAs for Grin1, Grin2a, Grin2b, Grin2c, Grin2d, and Rn28s1 were quantified by TAQMAN system using the following probes. The provider’s recommended pre-amplification step was performed for all the genes except for Rn28s1.
RNA levels were normalized by the quantity of Rn28s1, and standard curves were prepared for estimating the quantity (in femtograms) of the targeted RNAs after pre-amplification. For making the standard curves, total RNA from mouse brain hippocampal tissue was extracted using TRIZOL reagent. Target cDNAs were amplified by regular PCR, and amplicons were purified, quantity of cDNA was measured and submitted to serial dilutions, used for standard curves in TAQMAN system, in parallel to the single cell derived cDNA samples.
RNA levels were normalized by the quantity of Rn28s1, and standard curves were prepared for estimating the quantity (in femtograms) of the targeted RNAs after pre-amplification. For making the standard curves, total RNA from mouse brain hippocampal tissue was extracted using TRIZOL reagent. Target cDNAs were amplified by regular PCR, and amplicons were purified, quantity of cDNA was measured and submitted to serial dilutions, used for standard curves in TAQMAN system, in parallel to the single cell derived cDNA samples.
7
Single-Embryo qRT-PCR Analysis
8
Single-Cell qPCR Protocol for Gene Expression
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Single cells for qPCR were processed using the Single Cell-to-CT™ qRT-PCR Kit (Life Technologies) per manufacturer’s instructions. Cells were sorted directly into lysis solution in a 96-well plate, subjected to a reverse transcription reaction for cDNA synthesis, amplified for 14 cycles with pooled TaqMan Gene Expression Assays, and diluted with 1X TE buffer (pH 8.0). For real-time PCR, samples were amplified for 50 cycles of 3 seconds at 95°C followed by 30 seconds at 60°C using the following TaqMan probes: Mm00657672_m1 for Hoxb5, Mm03039759_s1 for Rnf208, Mm00470338_m1 for Smtnl1, and Mm99999915_g1 for Gapdh. All thermocycling was performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Presence of a single cell was validated by a CT value of 30 or less for Gapdh.
9
Single-Embryo qRT-PCR Analysis
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All single-embryo cDNA was prepared according to the Single Cell-to-Ct qRT-PCR kit (Life-Technologies, Cat# 4458236) with slight modifications. Pronuclear, 2-cell, 8-cell and blastocyst stage embryos were isolated and passed through three washes of PBS. Single embryos were then placed into individual PCR tubes and lysed in twice the recommended volume of Lysis/DNase (20 μL) for 15min at room temperature. Then, 2 μL of Stop Solution was added and incubated for 2 min. At this point, half of the reaction was stored in ‒80°C conditions as a technical replicate and the remaining sample (11 μL) continued through the original Single Cell-to-Ct protocol. All qRT-PCR reactions were performed using SSO Universal SYBR Green SuperMix, as per manufacturer instructions (Biorad, Cat# 1725275). All QPCR analyses were performed on the StepOnePlus Real Time PCR system (ThermoFisher, Cat# 437660).
10
Single-Cell Analysis of MERVL Expression
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