Total RNA was extracted from IECs, CD3+IELs, and challenged and sham-treated enteroids and tight monolayers by using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions and dissolved in RNase-free water containing rRNasin ribonuclease inhibitor (Promega, Madison, WI) and stored at -80°C until gene expression analysis by genome-wide hybridization bead array screening, real-time quantitative polymerase chain reaction array (qPCR-array) and/or qRT-PCR. The concentration of 18S rRNA was determined in each sample using real-time qRT-PCR (Applied Biosystems) in which triplicates of the samples were compared to triplicates of serial dilutions of a pool of total RNA extracted from polyclonally stimulated peripheral mononuclear cells [27 (link)]. Results are expressed as U/μL where one U was defined as the amount in 10 pg of the RNA standard.
Real time qrt pcr
Manufactured by Thermo Fisher Scientific
Real-time qRT-PCR is a laboratory instrument used for the quantitative analysis of RNA expression. It combines reverse transcription and real-time PCR techniques to measure and quantify specific RNA sequences in a sample.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Rrnasin ribonuclease inhibitor, by Promega (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
2 protocols using real time qrt pcr
1
Total RNA Extraction and Quantification
2
Quantification of Macrophage Markers in Cells
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Quantification of EMR1, CD206 and CD86 mRNAs was done using commercially available TaqMan Gene Expression Assays (EMR1: Hs00892591 m1, CD206: Hs00267207 m1; CD86: Hs01567026 m1) in combination with TaqMan EZ technology (Applied Biosystems). The RT-PCR profile was 50 • C for 2 min, 60 • C for 30 min and 95 • C for 5 min followed by 45 cycles of 95 • C for 20 s and 60 • C for 1min. Assays for CXCL16 and CXCL17 are previously described [10, 11] The concentration of 18S rRNA was determined in each sample by real-time qRT-PCR (Applied Biosystems) for normalization of mRNA levels as described [28] . All qRT-PCR analyses were performed in triplicates. Emission from the released reporter dye was recorded by the QuantStudio 5 Real-Time PCR System (Applied Biosystems) or the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems).
mRNA levels are expressed as relative quantity (RQ) where the mRNA concentrations are normalized to the 18S rRNA concentration in the same sample by calculating the CT between the CT for the mRNA species and the CT for 18S rRNA and RQ calculated according to the equation: 2 ∧ -( CT of the sample -the median CT value of the normal colon tissue samples).
mRNA levels are expressed as relative quantity (RQ) where the mRNA concentrations are normalized to the 18S rRNA concentration in the same sample by calculating the CT between the CT for the mRNA species and the CT for 18S rRNA and RQ calculated according to the equation: 2 ∧ -( CT of the sample -the median CT value of the normal colon tissue samples).
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