The RNA was isolated with the RNAeasy Kit (QIAGEN, USA) following reverse transcription to cDNA using a high-capacity cDNA transcription kit (QIAGEN, USA). The resulting cDNA libraries from each sample were then analyzed by qPCR for the genes Foxp3, IL-2, IL-10, IL-17, Tgfβ, Gzmb, Prf1, Ctla4, and interferon gamma. Gapdh was used as a housekeeping gene. The experiment was performed with 7500 v 2.6 software (Applied Biosystems, USA). Oligonucleotide primers are listed in Table S1 .
High capacity cdna transcription kit
Manufactured by Qiagen
Sourced in United States, Germany
The High-capacity cDNA Transcription Kit is a laboratory tool designed for the efficient conversion of RNA into complementary DNA (cDNA). It provides a reliable and streamlined method for generating high-quality cDNA from various RNA sources.
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3 protocols using high capacity cdna transcription kit
1
Quantifying Immune Gene Expression
2
Osteoclast Differentiation Assay with miR-CM and RANKL
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RAW264.7 cells (8×104 cells/well) were seeded in 12-well plates, and after 24 h, the medium was replaced with medium containing 10% of each miR-CM or 100 ng/ml of recombinant RANKL (PeproTech EC, Ltd.) as a positive control. Subsequently, 3 days after miR-CM or RANKL treatment, total RNA was isolated from RAW264.7 cells using the RNeasy mini kit (cat. no. 74104; Qiagen, Inc.) and cDNA synthesis was performed using the High-capacity cDNA Transcription kit (cat. no. 4368814; Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturers' protocol. Thereafter, mRNA expression of osteoclast differentiation markers, including nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), osteoclast-associated receptor (OSCAR), β3-integrin, cathepsin-K, and tartrate-resistant acid phosphatase (TRAP), in the cells was assessed by RT-qPCR (Fig. 1 ).
3
Quantitative Real-Time PCR for Gene Expression
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For the gene expression analysis by means of real time PCR, 2 × 105 fibroblasts per well were seeded in 6-well plates and cultivated to confluence. The drugs were added overnight in DMEM with 2% FBS. The next day, the total RNA was isolated using RNeasy Mini (Qiagen, Hilden, Germany) following manufacturer’s instructions. Using the High-Capacity cDNA Transcription Kit (Qiagen, Hilden, Germany), 1 μg of mRNA was transcribed into cDNA by following the manufacturer’s instructions. The relative gene expression analysis was performed on the ABI 7900H Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using the TaqMan Gene Expression Master Mix and the TaqMan probes according to the genes to be examined (Thermo Fisher Scientific, Waltham, MA, USA). The gene expression change was determined by the number of cycles at which a Threshold Cycle (Ct) was exceeded. The data thus obtained were normalized to an endogenous control and to each other with the untreated, non-diabetic control. Expression of the glo1 gene was quantified and normalized to gapdh as an endogenous control. The relative gene expression was calculated by the ΔΔCt method of Livak and Schmittgen (2001) [33 (link)].
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