Based on the array results, five types of miRNA were selected for further real-time PCR analysis. Real-time PCR was performed on RNA from the newly generated callus collected on post-fracture days five, seven, 11, 14, 21, and 28 (n = 6 in each group at each timepoint). Tissue specimens were homogenized and total RNA was extracted. RNA was reverse-transcribed into single-strand complementary DNA using the miRCURY locked nucleic acid (LNA) Universal RT microRNA PCR kit (Exiqon A/S, Vedbaek, Denmark). Real-time PCR analysis was performed in duplicate with a StepOne Sequence Detector (Applied Biosystems Inc., Foster City, California), using SYBR Green master mix and microRNA LNA PCR primer sets (both from Exiqon A/S, Vedbaek, Denmark). U6, a small nuclear RNA, was used as an internal control to normalize differences in miRNA levels in each sample.9 (link),12 (link) The relative abundance of each miRNA was calculated using the comparative ΔΔCT method9 (link),12 (link) and is presented as the fold change relative to levels in the post-fracture day five control sample.
Protocol detail
Temporal Profiling of Fracture-induced miRNA
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Callus
Fracture
Locked nucleic acid
Mirna
Primer
Real time pcr analysis
Rt pcr
Single strand dna
Small nuclear rna
Sybr green
Tissue
Corresponding Organization :
Other organizations : Kobe University, Showa University, Pain and Rehabilitation Medicine
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Variable analysis
independent variables
- Time points of callus collection (post-fracture days 5, 7, 11, 14, 21, and 28)
- Relative abundance of five selected miRNAs
- Tissue specimens were homogenized and total RNA was extracted
- RNA was reverse-transcribed into single-strand complementary DNA using the miRCURY locked nucleic acid (LNA) Universal RT microRNA PCR kit
- Real-time PCR analysis was performed in duplicate with a StepOne Sequence Detector, using SYBR Green master mix and microRNA LNA PCR primer sets
- U6, a small nuclear RNA, was used as an internal control to normalize differences in miRNA levels in each sample
- Positive control: The relative abundance of each miRNA was calculated using the comparative ΔΔCT method and is presented as the fold change relative to levels in the post-fracture day five control sample.
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