For cDNA synthesis and preamplification of target genes CellsDirect One-Step qRT-PCR kit (Invitrogen) was used. Cells were sorted directly into PCR plates or 0.2 mL PCR tubes containing 2.5μL gene specific 0.2x TaqMan gene expression assays (Applied Biosystems), 5μL of CellsDirect 2x Reaction mix (Invitrogen), 1.2μL CellsDirect RT/Taq mix, 1.2μL TE buffer and 0.1μL SUPERase-In RNase Inhibitor (Ambion) to a total volume of 10μL. Conditions for reverse transcription and target gene amplification were: 15 min at 50°C; 2min at 95°C; 22 cycles of 95°C for 15s and 60°C for 4min. Preamplified products were diluted 1:5 in TE buffer and analyzed on a 48x48 Dynamic Array (Fluidigm) using the following PCR cycling condition: 95°C for 10min; 40 cycles of 95°C for 15s and 60°C for 60s. Data was analyzed using the ΔΔCt method as described44 (link); results were normalized to B2m. Single cells not expressing Hprt or Actinb or B2m were excluded from the analysis. Taqman assays used for multiplexed quantitative real-time PCR are listed in Supplementary Table 4 .
48x48 dynamic array
Manufactured by Standard BioTools
The 48x48 Dynamic Array is a lab equipment product designed for high-throughput sample processing. It features a 48x48 grid layout that enables simultaneous processing of 2,304 individual samples or reactions. The device is engineered to provide precise and consistent results, making it a valuable tool for various applications in the scientific and research community.
Automatically generated - may contain errors
Lab products found in correlation
Cellsdirect 2x reaction mix, by Thermo Fisher Scientific (2 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Cellsdirect one step qrt pcr kit, by Thermo Fisher Scientific (2 mentions)
Cellsdirect rt taq mix, by Thermo Fisher Scientific (2 mentions)
2 protocols using 48x48 dynamic array
1
Single-Cell Gene Expression Analysis
2
Single-Cell Gene Expression Analysis
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For cDNA synthesis and preamplification of target genes CellsDirect One-Step qRT-PCR kit (Invitrogen) was used. Cells were sorted directly into PCR plates or 0.2 mL PCR tubes containing 2.5μL gene specific 0.2x TaqMan gene expression assays (Applied Biosystems), 5μL of CellsDirect 2x Reaction mix (Invitrogen), 1.2μL CellsDirect RT/Taq mix, 1.2μL TE buffer and 0.1μL SUPERase-In RNase Inhibitor (Ambion) to a total volume of 10μL. Conditions for reverse transcription and target gene amplification were: 15 min at 50°C; 2min at 95°C; 22 cycles of 95°C for 15s and 60°C for 4min. Preamplified products were diluted 1:5 in TE buffer and analyzed on a 48x48 Dynamic Array (Fluidigm) using the following PCR cycling condition: 95°C for 10min; 40 cycles of 95°C for 15s and 60°C for 60s. Data was analyzed using the ΔΔCt method as described44 (link); results were normalized to B2m. Single cells not expressing Hprt or Actinb or B2m were excluded from the analysis. Taqman assays used for multiplexed quantitative real-time PCR are listed in Supplementary Table 4 .
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