Cells were seeded in 24-well plates and stimulated in triplicate with or without 10 ng/mL recombinant human (rh) TGFβ1 for 1 week. Total RNA was purified using an RNeasy kit (Qiagen, Valencia, CA) according to manufacturer's instructions and quantitated by spectrophotometry with NanoDrop 2000c (Thermo Fisher Scientific). RNA was reverse transcribed using a high-capacity cDNA reverse transcriptase kit (Applied Biosystems, Foster City, CA) and subjected to TaqMan Gene Expression Assays (Applied Biosystems) for type I collagen (COL1A1, Hs00164004), α-SMA (ACTA2, Hs00426835), fibronectin (FN1, Hs01549940), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 4352665). PCR reaction was carried out in triplicate using TaqMan Fast Universal PCR Master Mix kit (Applied Biosystems) and 96-well optical plates on the StepOnePlus Real-Time PCR System (Applied Biosystems). The relative expression level of each mRNA transcript was normalized to GAPDH as an internal control as described previously18 (link).
Protocol detail
TGF-β1 Induced Fibrogenic Gene Expression
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RNeasy kit
NanoDrop 2000c
high-capacity cDNA reverse transcriptase kit
TaqMan Gene Expression Assays
TaqMan Fast Universal PCR Master Mix kit
StepOnePlus Real-Time PCR System
Acta2
Assays
Cdna
Cells
Gapdh
Gene expression
Glyceraldehyde 3 phosphate dehydrogenase
Human
Mrna
Real time pcr
Reverse transcriptase
Spectrophotometry
Tgfβ1
Type i collagen
Corresponding Organization : University of Pennsylvania
Other organizations : Seattle Children's Hospital, University Gastroenterology
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Variable analysis
independent variables
- Stimulation with or without 10 ng/mL recombinant human (rh) TGFβ1
- MRNA expression levels of type I collagen (COL1A1)
- MRNA expression levels of α-SMA (ACTA2)
- MRNA expression levels of fibronectin (FN1)
- Cell culture in 24-well plates
- RNA purification using RNeasy kit
- RNA quantitation by spectrophotometry with NanoDrop 2000c
- CDNA synthesis using high-capacity cDNA reverse transcriptase kit
- TaqMan Gene Expression Assays for target genes and GAPDH
- PCR reaction using TaqMan Fast Universal PCR Master Mix kit
- Real-time PCR on StepOnePlus system
- Normalization of mRNA expression to GAPDH as internal control
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