To interrogate osteogenic and proangiogenic gene expression, samples were collected in TRIzol Reagent (Invitrogen) for PCR analysis following the manufacturer’s instructions. After RNA isolation, 600 ng of RNA was reverse-transcribed with the QuantiTect Reverse Transcription Kit (QIAGEN, Valencia, CA), and qPCR was performed using the QuantiFast Probe PCR Kit (QIAGEN) on a QuantStudio 5 system (Applied Biosystems). Primers and probes for housekeeping genes RPL13 (Hs00744303_s1), RUNX2 (Hs01047973_m1), SP7 (Hs01866874_s1), BGLAP (Hs01587814_g1), IBSP (Hs00173720_m1), COL1A1 (Hs00164004_m1), BMP-2 (Hs00154192_m1), and VEGF-A (Hs00900055_m1) were purchased from Thermo Fisher Scientific. Amplification conditions were 95°C for 3 min, followed by 45 cycles at 95°C for 3 s and 60°C for 30 s. qPCR results were normalized to RPL13 transcript levels to yield ΔCt and cells from fresh BMA to yield ΔΔCt. Last, fold change in expression relative to the untreated and housekeeping gene was calculated using 2−ΔΔCt. Osteogenic and proangiogenic protein secretions were assessed by quantifying VEGF, OCN, and BMP-2 secretion with protein-specific ELISA kits per the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

RNA-seq library preparation was performed using Illumina TruSeq RNA Library Preparation Kit v2 (catalog no. RS-122-2002, Illumina), and single-end 75–base pair sequencing was performed using an Illumina NextSeq 500. Sequencing data quality was checked using FastQC software (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were mapped to the human genome (hg38) using STAR (49), and read counts per gene were determined using “featureCounts” from Rsubread package (50). Differentially expressed genes were identified using limma after voom normalization (51). A gene was considered as significantly differentially expressed when its false discovery rate–adjusted P value was <0.05 and fold change was >2. Gene ontology enrichment analysis was performed using ToppGene (52). Heatmaps were generated using the heatmap.2 function in “gplots” R package.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.