Cfx96 touch thermal cycler

Fracture calli were harvested 10 days post-fracture from β-catenin gain-of-function and control mice in order to assess Axin2 gene expression and confirm over-activation of the canonical Wnt pathway. Callus tissues were isolated from surrounding muscle and cortical bone and homogenized in TRIzol (Invitrogen, #15596026). RNA isolation was performed according to the manufacturer's protocol followed by DNase treatment (Invitrogen, #Am1906). cDNA was reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad, #1708890) and quantitative RT-PCR was performed using SYBRbased primers (Supplemental Table 1) and RT SYBR Green qPCR Mastermix (Qiagen, #330509) on a CFX96 Touch Thermal Cycler (Bio-Rad) run to 50 cycles (N=3-5/treatment group). RNA isolation, DNase treatment, and reverse transcription was performed as above for Wnt expression analysis on HUVEC cells. However, TaqMan probes (Supplemental Table 2) and TaqMan Universal Master Mix II with UNG (#4440038) were used. Analysis was run on a CFX96 Touch Thermal Cycler (Bio-Rad) to 45 cycles (N=3). All samples were run in triplicate and relative gene expression was calculated by normalizing to the housekeeping gene GAPDH (2 -△CT ). Fold change was calculated as 2 -△△CT .

Total RNA used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis was extracted from the second leaf of wheat (cv. Atlas 66) plants as described above. Total RNA (0.5 μg) was reversetranscribed in a total volume of 20 μl using the SensiFAST TM cDNA Synthesis Kit (Bioline). Real-time quantitative RT-PCR was performed on a CFX96 Touch TM Thermal Cycler (Bio-Rad) using the Luna ® Universal qPCR Master Mix (New England Biolabs). To quantify speci c transcripts from homoeologous genes, primers in Additional le 5: Table S2 were designed based on sequences retrieved from RNA-Seq data of cv. Atlas 66. For each gene, the reference gene sequence was retrieved from Ensembl Plants database and aligned to the assembled transcriptome generated by Trinity. The sequence of interest was identi ed based on the lowest E-value. Ampli cation was performed as follows: 5 min at 95°C followed by 35 cycles of 95°C for 15 sec, 58°C for 30 sec. The target gene transcript level was normalized against the 18S rRNA level. Fold changes of RNA transcripts were calculated using the 2 -ΔΔCt method [136].

Total RNA used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis was extracted from the second leaf of wheat (cv. Atlas 66) plants as described above. Total RNA (0.5 μg) was reverse-transcribed in a total volume of 20 μl using the SensiFAST TM cDNA Synthesis Kit (Bioline). Real-time quantitative RT-PCR was performed on a CFX96 Touch TM Thermal Cycler (Bio-Rad) using the Luna ® Universal qPCR Master Mix (New England Biolabs). To quantify specific transcripts from homoeologous genes, primers in Additional file 5: Table S2 were designed based on sequences retrieved from RNA-Seq data of cv. Atlas 66. For each gene, the reference gene sequence was retrieved from Ensembl Plants database and aligned to the assembled transcriptome generated by Trinity. The sequence of interest was identified based on the lowest E-value. Amplification was performed as follows: 5 min at 95°C followed by 35 cycles of 95°C for 15 sec, 58°C for 30 sec. The target gene transcript level was normalized against the 18S rRNA level. Fold changes of RNA transcripts were calculated using the 2 -ΔΔCt method [135] .