Computational protocol: Tendon proper- and peritenon-derived progenitor cells have unique tenogenic properties

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Protocol publication

[…] After 7 and 45 days in culture, total RNA was extracted from tendon proper- and peritenon-derived progenitor-seeded constructs by mechanically homogenizing tissues with a Tissue-Tearor (Biospec Products, Inc., Bartlesville, OK, USA) in QIAzol reagent (Qiagen, Valencia, CA, USA) and then using the QIAGEN RNeasy Plus Micro Kit []. Total RNA was isolated from cells in monolayer culture (pre-seeded cells compared to constructs or monolayer cultures for conditioned media studies) using the QIAGEN RNeasy Plus Micro Kit. For quantitative real-time polymerase chain reactions (RT-qPCR), total RNA (pre-seeded cells and tendon constructs, 250 ng; conditioned media monolayer culture, 200 ng) for each sample was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ABI, Foster City, CA, USA). This cDNA was used as a template for RT-qPCR with Fast-SYBR Green Master Mix (ABI). Assays were performed using the StepONEPlus Real-Time PCR System (ABI). Primers were designed using Primer Express software (ABI) for the following genes: actin, beta (Actb); scleraxis (Scx); tenomodulin (Tnmd); endomucin (Emcn); biglycan (Bgn); decorin (Dcn); collagen, type I, alpha 1 (Col1a1); collagen, type III, alpha 1 (Col3a1); collagen, type V, alpha 1 (Col5a1); collagen, type XI, alpha 1 (Col11a1); collagen, type XII, alpha 1 (Col12a1); and collagen, type XIV, alpha 1 (Col14a1). Primer sequences can be found in Table . Two technical replicates were performed for each biological replicate (n = 5 to 8 per group for tendon constructs; n = 4 to 8 for the conditioned media experiment). The results were adjusted for efficiency as measured by LinRegPCR using the default fit option that measures the slope of a line containing four to six data points and the highest R2 correlation value [,]. RT-qPCR data were normalized relative to the endogenous control gene, Actb. […]

Pipeline specifications

Software tools Primer Express, LinRegPCR
Application qPCR