qPCR was performed using Taqman low density array (TLDA) microfluidic cards with 48 targets per sample (48a format) (Applied Biosystems) and QuantiTect primer assays (Qiagen) with Power SYBR green fluorescent reporter (Applied Biosystems). Details are given in the Supplementary material online , Methods.
Taqman low density array microfluidic cards
Manufactured by Thermo Fisher Scientific
The Taqman low density array (TLDA) microfluidic cards are a high-throughput gene expression analysis tool. They enable the measurement of multiple gene targets simultaneously in a single sample. The cards are pre-loaded with gene-specific Taqman assays and allow for the rapid and efficient quantification of gene expression levels.
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Lab products found in correlation
Quantitect, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Dataassist software, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Prism 7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
4 protocols using taqman low density array microfluidic cards
1
Comprehensive qPCR Using TLDA Cards
2
Tumor-Metastasis-Related Gene Expression Analysis
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The SK-MEL-5 cells (2 × 105 cells/mL) were treated with DMSO or PG at 0.5 µM for 16 h and RNA extraction was performed with RNeasy® mini kit (Qiagen, Valencia, CA, USA). In order to obtain the cDNA, 1 µg RNA was used for reverse transcription with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster, CA, USA). TaqMan® Low-Density Array (TLDA) micro fluidic cards (Applied Biosystems), designed for assessing the expression of 87 known human tumor-metastasis-related genes and 8 endogenous controls, were used. The experiment was then performed according to manufacturer’s instructions using the 7900HT Fast Real-Time PCR system (Applied Biosystems). Each sample was run in duplicate. The DataAssistTM software (Applied Biosystems) was used to determine the most appropriate endogenous control gene for our experimental conditions and to analyze the results. Beta-2-microglobulin was found to be the most stable gene after PG treatment.
3
miRNA Profiling of C14MC Cluster
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miRNA profiling for the C14MC microRNA cluster was performed using customized TaqMan low density array (TLDA) microfluidic cards (Applied Biosystems, CA) as described earlier [23 (link)]. Details of the candidate miRNAs and endogenous controls found on TLDA cards are provided in Supplementary Table S3. Threshold cycle (Ct) values greater than 35 were imputed to 35 according to the technical recommendation [24 ]. The Ct was calculated by relative quantification using 2−∆∆Ct method [18 (link)].
4
Chemokine Expression Profiling Using TLDA
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Chemokine transcription was assessed using custom-made TaqMan low-density array (TLDA) microfluidic cards (Applied Biosystems, Foster City, CA) as described previously [20 (link)]. Briefly, 100 μl of reaction mix, containing a 1:1 mixture of cDNA (from ~ 1 μg total RNA) in RNase-free water and 2x TaqMan Universal PCR Master Mix (Thermo Fisher Scientific, Waltham, MA), was loaded onto cards containing the primers and probesets for 32 genes. Cards were centrifuged to distribute the reaction mixture throughout the fluidic system and run on a Prism 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA). Data were analysed using SDS2.2 software and RQ Manager. Relative gene expression was first normalised to the expression of housekeeping gene Tbp, which encodes TATA-binding protein, and then normalised to that of a calibrator selected arbitrarily from the vehicle control group. Fold change of gene expression in LPS-challenged mice compared to vehicle controls was calculated using the ΔΔCT method.
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