For whole bone gene expression, calvaria were cleaned of soft tissue, flash frozen, and homogenized in TRIzol reagent (Life Technologies); RNA was extracted as directed by the manufacturer. As previously described, total RNA was quantified, DNase treated (Promega), and subjected to random hexamer primed reverse transcription for Sybr green based quantitative PCR (qPCR) (4 (link)). For Sybr green based qPCR, gene expression was normalized to 18S rRNA. Primer sets are shown in Supplemental Table 1 . miRNA levels were quantified using miRNA-specific TaqMan microRNA assays (Life Technologies). Briefly, miRNAs were reverse transcribed from total RNA using target-specific primers to create cDNA, which was quantified by qPCR. miRNA expression was normalized to RNU6B. Relative quantities were determined using standard curves and samples were assayed in duplicate. RNA from each mouse (biological replicate) was analyzed in duplicate (technical replicate).
Mirna specific taqman microrna assays
Manufactured by Thermo Fisher Scientific
The MiRNA-specific TaqMan microRNA assays are a set of laboratory reagents designed for the detection and quantification of specific microRNA (miRNA) molecules. These assays utilize TaqMan technology, a PCR-based method, to accurately measure the expression levels of individual miRNA targets.
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2 protocols using mirna specific taqman microrna assays
1
Quantifying Bone Gene Expression
2
Plasma miRNA Extraction and Quantification
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Prior to any therapeutic procedures, 1 ml of whole blood from each participant was collected into EDTA tubes after overnight fasting. Samples were immediately centrifuged at 3000 g for 10 min at room temperature, then centrifuged at 10,000 g for 5 min at 4°C, and subsequently stored at −80°C until analysis. Samples were frozen within 3 h of plasma separation.
After two subsequent spins, total RNA was extracted from 100 μl of plasma using an RNA purification kit (Norgen Biotek Corporation, Thorold, ON, Canada) that isolates enriched miR species. The synthetic Caenorhabditis elegans miR, cel-miR-39, was spiked into plasma before RNA extraction. Only samples with cel-miR-39 recovery > 95 % were used in subsequent analyses.
MiRNAs were reverse-transcribed with the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, United States) as recommended by the manufacturer. MiRNA-specific TaqMan MicroRNA Assays (Life Technologies) were used for plasma miRNAs.
Data were analysed by the 2−ΔCt method. Relative expression corresponded to the 2−ΔCt value. For analysis of miRNA expression levels, external normalization to cel-miR-39 was applied.
After two subsequent spins, total RNA was extracted from 100 μl of plasma using an RNA purification kit (Norgen Biotek Corporation, Thorold, ON, Canada) that isolates enriched miR species. The synthetic Caenorhabditis elegans miR, cel-miR-39, was spiked into plasma before RNA extraction. Only samples with cel-miR-39 recovery > 95 % were used in subsequent analyses.
MiRNAs were reverse-transcribed with the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, United States) as recommended by the manufacturer. MiRNA-specific TaqMan MicroRNA Assays (Life Technologies) were used for plasma miRNAs.
Data were analysed by the 2−ΔCt method. Relative expression corresponded to the 2−ΔCt value. For analysis of miRNA expression levels, external normalization to cel-miR-39 was applied.
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