Total RNA was isolated using the Direct-zol RNA Kit (Zymo Research, Germany) according to the manufacturer’s instructions. Human lung tissue was transferred to Lysing Matrix D tubes (MP Biomedicals, Germany) in 500 μl TRIzol reagent. The tissue was lysed using a FastPrep-24 device (MP Biomedicals) by applying four rounds of tissue lysis at default settings (4 m/s, 20 s). Cells were directly lysed in 500 μl TRIzol reagent. The homogenates were centrifuged and 0.5 µg of RNA purified from the supernatant was reverse transcribed for the profiling of IFN subtypes using the RT2 Profiler Kit (Qiagen, Germany).
Rt2 profiler kit
Manufactured by Qiagen
Sourced in Germany
The RT2 Profiler Kit is a qPCR array designed for the analysis of gene expression profiles. The kit provides a comprehensive and accurate way to simultaneously measure the expression levels of a focused panel of genes related to a specific biological pathway or disease state. The core function of the RT2 Profiler Kit is to enable reliable and reproducible gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Cfx manager software, by Bio-Rad (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Direct zol rna kit, by Zymo Research (1 mentions)
Lysing matrix d tube, by MP Biomedicals (1 mentions)
Fastprep 24 device, by MP Biomedicals (1 mentions)
Rt2 first strand kit, by Qiagen (1 mentions)
Sybr green 1 reagent, by Takara Bio (1 mentions)
Lc480 platform, by Roche (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Rneasy midi kit, by Qiagen (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Fastlane cdna analysis kit, by Qiagen (1 mentions)
5 protocols using rt2 profiler kit
1
RNA Extraction and cDNA Synthesis
2
Liver Cancer Transcriptome Analysis Protocol
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Total RNA was extracted using RNeasy mini kit (Qiagen, Germany) under manufacturer's instruction. First strand cDNA was prepared with RT2 First Strand Kit (Qiagen, Germany). For PCR array, the pre-coated Liver Cancer RT2 Profiler kit (PAHS-133Z, Qiagen, Germany) was used; for real-time PCR, cDNA was amplified in SYBR Green I reagent (Takara, Japan) with specific primer sets (Supplementary Table 1 ) for particular genes. All the assays were performed on LC480 platform (Roche, USA).
3
Quantitative Analysis of Breast Cancer EMT
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We used an RNEasy mini kit (cat#74104, Qiagen, MD, USA) to isolate total RNA from MDA231 cells of all experimental groups. A QuantiTect Reverse Transcription Kit (cat# 205313, Qiagen, MD, USA) was used to transcribe RNA into the single stranded cDNA. Real-time PCR reaction was performed to detect the expression of 84 genes related to Human Breast Cancer using an RT2 Profiler Kit (cat# PAHS-131ZA, Qiagen, Hilden, Germany) and the RT² Profiler PCR Array Human Epithelial to Mesenchymal Transition (Qiagen Cat# PAHS-090ZA) to analyze modulation in the expression of genes responsible for EMT on the CFX96 real-time PCR detection system (Bio-Rad, CA, USA). Data analysis was performed using the CFX manager software (Bio-Rad, CA, USA). The data were analyzed by calculating ΔΔ−2 values and expressed as fold change expression, as compared to the relative expression of GAPDH used as a loading control and the untreated MDA231 cells as an experimental control. Experiments were repeated three times using CVMSCs isolated from five different placentas.
4
Profiling Inflammatory Cytokines in Rabbit Lungs
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Right lungs were harvested, cut into <0.5-cm pieces, and preserved in RNAlater (Thermo Fisher Scientific) immediately after euthanasia. RNA was extracted using the DNase treatment option with the RNeasy midi kit (Qiagen), followed by cDNA synthesis according to the manufacturer's instructions. Gene expression profiles were then evaluated using the rabbit Inflammatory Cytokines & Receptors Real-Time Reverse Transcriptase (RT2)-Profiler kit (Qiagen), according to the manufacturer's instructions. Each RT2 Profiler was performed twice per animal, which included evaluation of a technical replicate. The RNA integrity number (RIN) evaluation (>5), PCR array reproducibility, reverse transcriptase efficiency, and genomic DNA contamination were evaluated for each RNA extraction for quality control purposes. Average cycle threshold (CT) values were used for each tissue, with a threshold of 0.051. The mean CT values of four rabbit housekeeping genes (HKGs), ACTB, GAPDH, LDHA, and loc100346936, were used for normalization. For each gene, the fold regulation was evaluated as fold change of the MEDI3902 treatment group relative to the c-IgG treatment group using normalized CT values.
5
Quantifying Cytokine and Chemokine Genes
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We used RNEasy mini kit (cat#74104, Qiagen, MD, United States) to isolate total RNA from treated and untreated cells. Fastlane cDNA Analysis Kit (cat#215011, Qiagen, MD, United States) was used to transcribe RNA into the single-stranded cDNA. Real-time PCR reaction was performed to detect the expression of 84 genes related to Human Cytokines & Chemokines using a RT2 Profiler Kit (cat#PAHS-150Z, Qiagen, Hilden, Germany) on the CFX96 real-time PCR detection system (Bio-Rad, CA, United States). Data were initially analyzed using the CFX manager software (Bio-Rad, CA, United States) and for further analyses were exported to Microsoft Excel. The data were analyzed by calculating ΔΔ–2 values, and expressed as fold change expression as compared to the relative expression of house-keeping genes used as a loading control. Experiments were performed in triplicate and repeated three times using CV-MSCs prepared from three independent placentae.
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