Single cell to ct kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Single Cell-to-CT kit is a laboratory tool designed for the preparation and analysis of single-cell samples. It provides a streamlined workflow for isolating and processing individual cells to enable downstream molecular analysis, such as gene expression profiling.

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Lab products found in correlation

Biomark hd system, by Standard BioTools (3 mentions) Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions) Ssofast evagreen supermix with low rox, by Bio-Rad (2 mentions) Taqman assay, by Thermo Fisher Scientific (2 mentions) Biomark system, by Standard BioTools (2 mentions) C1 single cell auto prep reagent kit, by Standard BioTools (2 mentions) C1 single cell auto prep system, by Standard BioTools (2 mentions) Real time pcr analysis, by Standard BioTools (1 mentions) Rt2 first strand kit, by Qiagen (1 mentions) Picopure rna isolation kit, by Arcturus (1 mentions) 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions) Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions) Quantstudio 7 flex real time pcr machine, by Thermo Fisher Scientific (1 mentions) Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Collagenase 2, by Worthington (1 mentions) Collagenase p, by Roche (1 mentions) Dispase, by Worthington (1 mentions) C1 auto prep platform, by Standard BioTools (1 mentions) Taqman real time pcr assay, by Thermo Fisher Scientific (1 mentions) Rmwnt3a, by R&D Systems (1 mentions)

32 protocols using single cell to ct kit

Single-Cell qPCR for Gene Expression

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Single cell qPCR was performed as previously described (Nefzger et al., 2016 (link)). In brief, cells were flow
cytometry deposited into qPCR 96-well plates filled with 10μL of lysis
buffer and processed with the Single Cell to C_T kit (#4458236, Thermo
Fisher). cDNA was produced from the lysate as per kit’s instructions.
Samples were submitted to 18 cycles of pre-amplification using the following
TaqMan assays (Thermo Fisher): Actb (Mm00607939_s1),
Foxo1 (Mm00490672_m1), c-Myc(Mm00487804_m1), Apex1 (Mm01319526_g1), Apex2(Mm00518685_m1), Prdm1 (Mm00476128_m1), Bcl6(Mm00477633_m1), Aicda (Mm01184115_m1), Cxcr4(Mm01292123_m1), and Cd86 (Mm00444543_m1). GLTs were detected
using custom probe sets (see Table S1). Single cell qPCR data collection was performed with a
Biomark instrument (Fluidigm) on pre-amplified templates that were positive
screened for the housekeeper Actb (manually tested by qPCR).
Reactions were run for 40 cycles. Data was analyzed using the Biomark software
package “Real-Time PCR analysis” (Fluidigm). Data cleaning and
normalization were done using custom R code (R version 3.3.3, R Core Team). The
limit of detection was set to 40 cycles. Undetermined C_T were given a
value of 40. Heatmaps and violin plots of the resulting data were generated
using the ggplot2 package (version 2.2.1) in the R environment.

Roco J.A., Mesin L., Binder S.C., Nefzger C., Gonzalez-Figueroa P., Canete P.F., Ellyard J., Shen Q., Robert P.A., Cappello J., Vohra H., Zhang Y., Nowosad C.R., Schiepers A., Corcoran L.M., Toellner K.M., Polo J., Meyer-Hermann M., Victora G, & Vinuesa C.G. (2019). Class Switch Recombination Occurs Infrequently in Germinal Centers. Immunity, 51(2), 337-350.e7.

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Quantitative RT-PCR for FACS Sorted Cells

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RNA for the FACS sorted cells was extracted using the Pico pure RNA isolation kit (Arcturus kit0204). Purification was performed according to the manufacturer's protocol using RNA extraction from cell pellet protocol with on-column DNase treatment and eluted with 12 µL of elution buffer. cDNA was synthesized from 40 ng of isolated RNA using the RT² first strand kit (Qiagen 330404). Preamplification of cDNA was done using single cell to CT kit (Thermo Fisher Scientific 445 8237) and TaqMan gene expression assay primer sets (gapdh [Mm99999915_g1], nr3c1 [Mm00433832_m1], and nr3c2 [Mm01241596_m1]). Standard qRT-PCR was done using TaqMan 2× universal mix (ABI 430 4437) with same primer sets on an Applied Biosystem 7500 Fast real-time PCR system.

Taylor W.W., Imhoff B.R., Sathi Z.S., Liu W.Y., Garza K.M, & Dias B.G. (2021). Contributions of glucocorticoid receptors in cortical astrocytes to memory recall. Learning & Memory, 28(4), 126-133.

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Single-Cell qPCR Profiling Protocol

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The single oocytes were prepared using the Single Cell-to-Ct Kit (ThermoFisher Scientific) following the manufacturer’s instructions and loaded onto a TAC for qPCR. The reverse transcription and preamplification steps were conducted in a clean PCR workstation. After pre-amplification, the cDNA was diluted with 1× TE buffer at 1:20. The TACs were run in a QuantStudio 7 Flex Real-Time PCR machine (ThermoFisher Scientific), which contains a TaqMan array block. The RNA from pooled cells was reverse transcribed using a SuperScript VILO cDNA Synthesis Kit (ThermoFisher Scientific), and qPCR was performed with the Power Up SYBR Green Master Mix (ThermoFisher Scientific) following the manufacturer’s instructions. The qPCR primer sequences are shown in Table S2.

Lv Y., Cao R.C., Liu H.B., Su X.W., Lu G., Ma J.L, & Chan W.Y. (2021). Single-Oocyte Gene Expression Suggests That Curcumin Can Protect the Ovarian Reserve by Regulating the PTEN-AKT-FOXO3a Pathway. International Journal of Molecular Sciences, 22(12), 6570.

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Isolation and Analysis of Suture-Derived Cells

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Four-week-old Prx1-creER-EGFP^+/− or Col1-EGFP^+/− mice were euthanized for EGFP⁺ cell isolation from coronal and sagittal sutures using FACS. Coronal and sagittal sutures were dissected, pooled, and digested at 37°C in a shaking water bath in two steps. First, with 3 mg/mL of collagenase II (Worthington Biochemical; cat. #LS004176) in αMEM and 1% penicillin/streptomycin for 90 min; then with 0.76 U/mL of collagenase P (Roche; cat. #11249002001), 0.67 U/mL of dispase (Worthington Biochemical; cat. #LS02104) in αMEM, and 1% penicillin/streptomycin for 30 min.
EGFP⁺ and EGFP⁻ cells were sorted (average of 10,000). Cell lysis, RNA isolation, and reverse transcription for gene expression analysis were performed using the Single Cell to CT kit (Thermo Fisher Scientific; cat. #4458237). qRT-PCR reactions were carried out using the TaqMan gene expression analysis technology. TaqMan assay codes for each gene primer set are listed in Table 1 (n = 3 independent experiments).

Wilk K., Yeh S.C., Mortensen L.J., Ghaffarigarakani S., Lombardo C.M., Bassir S.H., Aldawood Z.A., Lin C.P, & Intini G. (2017). Postnatal Calvarial Skeletal Stem Cells Expressing PRX1 Reside Exclusively in the Calvarial Sutures and Are Required for Bone Regeneration. Stem Cell Reports, 8(4), 933-946.

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Molecular Profiling of iPSC-derived Cells

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Single iPSCs, NPCs and MNs were captured on C1 auto prep platform (Fluidigm, CA). All non-single cells were discarded from analysis. cDNA from single cells were prepared using the Single-Cell-to-Ct kit (ThermoFisher, USA) and pre-amplified with a pool of primers designed for the splicing events and the expression of corresponding genes (Table S1). Inclusion and exclusion primers were specifically designed to quantitate inclusion and exclusion of AS exons and expression primers were designed from constitutive exons. All primers were tested for amplification efficiency. High-throughout quantitative PCR was performed on 96.96 Dynamic Arrays on BioMark system (Fluidigm) according to manufacturer’s instructions. 3 housekeeping genes (RPL22, RPL27, PGK) and lineage genes (POU5F1, LIN28A, DPPA2, PAX6, NES, ISL1, MNX1, STMN2) were included.

Song Y., Botvinnik O.B., Lovci M.T., Kakaradov B., Liu P., Xu J.L, & Yeo G.W. (2017). Single-cell alternative splicing analysis with Expedition reveals splicing dynamics during neuron differentiation. Molecular cell, 67(1), 148-161.e5.

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Studying Wnt Signaling in Bone Cells

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For in vitro studies, EGFP⁺ and EGFP⁻ cells were sorted by FACS from calvaria of newborn animals (n = 5–7 animals) and seeded into 6-well plates in basic medium (αMEM, 20% FBS, 1% penicillin/streptomycin) for cell culture. Cells (passage 4) were treated with either 50 ng/mL of rmWNt3a or 1,000 ng/mL of rmDDK1 (R&D Systems) and incubated for 48 hr, or pretreated for 24 hr with 1,000 ng/mL of rmDKK1 (R&D Systems) and then stimulated with 50 ng/mL of rmWNt3a (R&D Systems) for 48 hr. qRT-PCR was performed using TaqMan real-time PCR assays (Life Technologies) (n = 3 independent experiments). For in vivo studies, 7-day-old Prx1-creER-EGFP^+/− animals received calvarial subperiosteal injections of rmWNt3a (440 ng/day in PBS, R&D Systems) (n = 6 mice) or control (PBS) (n = 5 mice) for 2 consecutive days. After 24 hr, EGFP⁺ cells were isolated from calvarial bones by FACS and qRT-PCR was performed using the Single Cell to CT kit (Thermo Fisher Scientific; cat. #4458237).

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Single-Cell Multiplexed qRT-PCR Analysis

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For single-cell multiplexed qRT-PCR (Figures S1A andS1B), preamplified cDNA templates were prepared from FACS-sorted fractions from GFRα1-EGFP mice. The sorted single cells (a 10:1 mixture of EGFP + E-Cadherin + CD9 + KIT -/low and EGFP -E-Cadherin + CD9 + KIT -/low A undiff fractions) were captured individually on a C1 Single-Cell Auto Prep Array for PreAmp (Fluidigm) designed for 10-to 17-um cells using a Fluidigm C1 system (Fluidigm). Cell lysis, cDNA synthesis, and preamplification were performed using Single Cell-to-CT Kit (Thermo), C1 Single-Cell Auto Prep Reagent kit, and specific primers for arbitrarily selected target genes (Table S2). qPCR were performed on a 96:96 dynamic array chips (Fluidigm) using GE 96.96 Dynamic Array DNA Binding Dye Sample & Loading Reagent Kit (Fluidigm), and SsoFast EvaGreen Supermix with Low ROX (Bio-Rad), in a Biomark HD system (Fluidigm) platform, following the manufacturer's instructions. The Raw data from 60 cells were analyzed in RStudio software (https://www.rstudio.com/). The detection limit of Ct value was set to 24; results are expressed as 2 (24 -Ct) .

Nakagawa T., Jörg D.J., Watanabe H., Mizuno S., Han S., Ikeda T., Omatsu Y., Nishimura K., Fujita M., Takahashi S., Kondoh G., Simons B.D., Yoshida S, & Nagasawa T. (2021). A multistate stem cell dynamics maintains homeostasis in mouse spermatogenesis. Cell reports, 37(3).

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Single-cell qRT-PCR of Trpa1, Trpv1, and Gapdh

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Single-cell qRT-PCR was performed with Invitrogen Single Cell-to-CT kit (4458237, Invitrogen) according to the manufacturer’s manual. TaqMan assays were used to measure the abundance of Trpa1 (TaqMan assay Mm01227437_m1), Trpv1 (TaqMan assay Mm01246300_m1), and Gapdh (TaqMan assay Hs02758991_g1).

Xie Z., Feng J., Cai T., McCarthy R., Eschbach MD I.I., Wang Y., Zhao Y., Yi Z., Zang K., Yuan Y., Hu X., Li F., Liu Q., Das A., England S.K, & Hu H. (2022). Estrogen metabolites increase nociceptor hyperactivity in a mouse model of uterine pain. JCI Insight, 7(10), e149107.

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Single-cell gene expression analysis

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Single tdTom⁺ MEFs were sorted into a 96-well plate containing cell lysis buffer. Reverse transcription and preamplification of genes of interest were performed using the Single-Cell-to-CT Kit (Invitrogen) according to the manufacturer’s guidelines, followed qRT-PCR analysis, as described above.

Liu J.Y., Souroullas G.P., Diekman B.O., Krishnamurthy J., Hall B.M., Sorrentino J.A., Parker J.S., Sessions G.A., Gudkov A.V, & Sharpless N.E. (2019). Cells exhibiting strong p16INK4a promoter activation in vivo display features of senescence. Proceedings of the National Academy of Sciences of the United States of America, 116(7), 2603-2611.

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Quantifying mRNA Expression via RT-qPCR

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The mRNA was isolated by adding 100 ul of the lysis buffer without DNase I (Single Cell-to-CT kit, Invitrogen 4458237) directly to the sorted cells or the adherent cells on dish. After incubating at 37C for 20 min, the digestion was stopped by adding stop buffer. Fifteen µL of the lysates were used as the templates for reverse transcription with MMLV reverse transcriptase (Enzymatics) and Oligo d(T)23VN. The cDNA was used for SYBR-based RT-qPCR assays (supplementary Table 1). The fluorescence data was analyzed by “qpcR” package with the following parameters: baseline = 1:8, norm = TRUE, methods = “sigfit”, model = l5, type = “Cy0”, which.eff = “sig”, type.eff = “mean.pair”, which.cp = “Cy0”57 (link). The means and standard deviations from permutation analysis were exported for further statistical analysis.

Wu Y.T., I.-Shing Y.u., Tsai K.J., Shih C.Y., Hwang S.M., Su I.J, & Chiang P.M. (2015). Defining Minimum Essential Factors to Derive Highly Pure Human Endothelial Cells from iPS/ES Cells in an Animal Substance-Free System. Scientific Reports, 5, 9718.

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