Superscript 3 reverse

At 1, 3, and 7 days after ligature placement, total RNA was extracted from the harvested ligature side or non-ligature side of the maxillary gingival tissues with the RNeasy Mini Kit (QIAGEN, Germantown, MD) and quantified with a Thermo Scientific NanoDrop 1000 ultraviolet-visible spectrophotometer (NanoDrop Technologies, Wilmington, DE). After treatment with DNase I (Thermo Fisher Scientific), cDNA was synthesized from 1 µg of total RNA using Super Script III reverse transcriptase (Super Script VILO: Thermo Fisher Scientific).
Taqman-based qRT-PCR was performed using commercially available primer/probe mixes as follows, Il1b (Mm00434228_m1, Thermo Fisher Scientific), Il6 (Mm00446190_m1, Thermo Fisher Scientific), Il17a (Mm00439618_m1, Thermo Fisher Scientific) and Tnfsf11 (RANKL) (Mm00441908_m1, Thermo Fisher Scientific) in combination with a mouse Gapdh internal control mix (Mm99999915_g1, Thermo Fisher Scientific). Target gene expression was quantitatively analyzed using the ΔΔCT method. Statistical analysis was performed using Student’s t test to assess the difference between the ligature side group and the non-ligature side group at each time point. P < 0.05 was considered as statistically significant.

Total RNA was extracted using TRIzol™ (Thermo Fisher Scientific, Waltham, MA, USA, Cat#15596026) as instructed by the manufacturer. Complementary DNA was subsequently synthesized from total RNA using SuperScript™ III reverse transcriptase (Thermo Fisher Scientific, Cat#18080093) and PCR was performed using a PCR thermal cycler (Eppendorf, Germany, MasterCycler epgradients). The PCR program used for amplification consisted of 2 min at 92 °C, followed by 35 cycles of 5 s at 92 °C, 10 s at 60 °C, and 20 s at 72 °C and concluded with 10 min at 72 °C. Beta-2-Microglobulin (B2M) gene was used to analyse the relative gene expression with either the dCt or the ddCt method. All the primer sequences are listed in Supplementary Table S3.

Total RNA was extracted as previously described (Box et al. 2011 (link)). Genomic DNA was digested with TURBO DNA-free (Ambion Turbo DNase kit AM1907) according to the manufacturer's guidelines, before reverse transcription was performed. The reverse transcription was performed with the SuperScript III reverse transcriptase (ThermoFisher 18080093) following the manufacturer's protocol using gene-specific primers. Relevant primers are listed in Supplemental Tables S3 and S4. For field experiments, RNA extraction and QPCR were performed as described (Antoniou-Kourounioti et al. 2018 (link); Hepworth et al. 2018 (link), 2020 (link)). In brief, analysis of qPCR results was performed with LinReg with normalization to the geometric means of the At5g25760 (“PP2A”) and At1g13320 (“UBC”) control genes. The same analysis was also used in Figure 1, C–E, and Supplemental Figure S3, C–E. The rest results were normalized to single reference gene, UBC. Primers used are described in Supplemental Table S3.