Cfx384 real time system machine

Total RNA from flushed and minced femoral bones or cell cultures was isolated using the Direct-Zol RNA Mini Prep Kit (Zymo Research) following the supplier’s instructions. For quantitative real-time polymerase chain reaction (rt-qPCR), cDNA was synthesized from ∼0.5 μg RNA using the SuperScript III first strand synthesis system (Invitrogen) as described by the supplier and subjected to rt-qPCR amplification using the QuantiTect SYBR-Green PCR Kit (Qiagen) and a CFX384 real time system machine (Bio-Rad) using the following conditions: 10 min 95 °C followed by 45 cycles of 30 sec at 95 °C, 30 sec at 60 °C and 30 s at 72 °C. Primers for the analyzed genes are listed in Table S1. All rt-qPCR assays were performed in triplicate and expression was evaluated using the Biorad CFX Manager 3.0 software applying the comparative quantification method^{75 (link)}. For high through output next generation RNA sequencing (RNA-seq) RNA integrity was analyzed with a 2100 Bioanalyzer with an RNA 6000 kit (Agilent Technologies). RNA-Seq libraries were prepared using the Illumina TruSeq v2 library preparation kit and sequenced with paired-end 50 bp reads on an Illumina HiSeq 4000 in biological triplicates for bone tissues (femurs) and duplicates for cell-lines.

RNA was isolated using the miRNeasy kit (Qiagen, Hilden, Germany). Isolated RNA was reverse transcribed into cDNA using the SuperScript III first strand synthesis system (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Gene expression for selected gene markers was quantified using RT-qPCR whereby each reaction was performed with 2.5 ng of cDNA per 10 ul, QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany), and the CFX384 real time system machine (Bio-Rad, Hercules, California, USA). Transcript levels were quantified using the 2^ΔΔCt method and normalized to the housekeeping gene Actb (set at 100).

Total RNA was isolated using the Direct-zol™ RNA kit (Zymo Research). Before isolation, calvaria (2-to-3-day old mice) and other tissues (eight-week-old mice) were homogenized for 30 s in ice-cold QIAzol using the Ultra Turrax T25 tissue homogenizer (IKA). RNA was quantified using the NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific).
For mRNA analysis, RNA was reverse transcribed into cDNA by the SuperScript III First-Strand Synthesis System (Invitrogen). Gene levels were assessed using real-time PCR using the QuantiTect SYBR Green PCR Kit (Qiagen) and the CFX384 Real-Time System machine (BioRad). Transcript levels were quantified using the 2^ΔΔCt method and normalized to the housekeeping gene Gapdh (set at 100). Primer pairs are shown in Suppl Table 3.
For miRNA quantification, miRNAs were reverse transcribed into cDNA using the Mir-X miRNA First-Strand Synthesis Kit as instructed (Takara). Similar to mRNA, miRNA levels were determined using QuantiTect SYBR Green PCR Kit and the CFX384 Real-Time System machine. miRNA quantification was assessed using a universal reverse primer provided in First-Strand Synthesis Kit (Takara) and miRNA specific forward primers listed in Suppl Table 3. As with mRNA, miRNA levels were quantified using the 2^ΔΔCt method and normalized to miR-103a-3p (set at 100)^{71 (link)}.

RNA was extracted using TRIzol (Invitrogen) based on the manufacturer's instructions. DNA was removed with DNase I (New England Biolabs) and RNA was purified using Monarch® RNA Cleanup Kit (New England Biolabs). Complementary DNA (cDNA) was generated using High‐Capacity RNA‐to‐cDNA Kit (Applied Biosystems) and the cDNAs were diluted 10‐fold prior to qPCR measurements. The qPCR assay was performed with iTaq Universal SYBR Green Supermix (BioRad) on CFX384 Real‐Time System machine (BioRad). qPCR primers are listed in Appendix Table S1. All experiments were performed in technical and biological triplicates. Primer efficiency and qPCR quantification were analyzed as previously described (Siwek et al, 2020 (link)).