Sybr green 1 dye

Total RNA was extracted from mouse liver tissue with TRIzol (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was performed with Superscript III reverse‐transcription reagent (Invitrogen) using 1 μg RNA. Quantitative real‐time PCR was conducted with SYBR Green I dye (Roche, Basel, Switzerland). PCR cycling started at 95°C for 30 seconds followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, with the last step at 72°C for 20 min. The primer sequences used were listed in our previous study.⁹ Relative mRNA level changes were analyzed using the 2^−ΔΔCt method.

Following primer design with Primer3 [21 (link)], the expression levels of 17 selected genes were defined by qPCR to validate the RNA-seq results and evaluate time-related expression trends (Table 2). The specificity of each primer pair was preliminary verified with BLAST searches.
Briefly, 1 μg of total RNA was retro-transcribed into cDNA using oligo(dT)_12–18, 25 ng primers and 200 U of SuperScript II Reverse Transcriptase (Life Technologies). Quantitative PCR analysis was carried out with the Rotor Gene RG-3000A Real Time PCR system (Corbett Research, Sydney, Australia) using the SYBR Green I dye (Roche, Mannheim, Germany) and combined sense and antisense primers at 300 nM final concentration. We used the following cycling conditions: an initial denaturation step of 10 min at 95°C, 45 cycles of amplification consisting of 15 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C. cDNA samples were analyzed in triplicate. Gene expression was evaluated with ΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. GAPDH was identified also by sequencing data as the most stably expressed housekeeping gene in corneal samples [22 (link)]. Results are expressed as gene expression fold change of the treated corneas compared to the control ones.

cDNA was reverse-transcribed from 8.0 µg total RNA using M-MLT RT (Invitrogen Corp., Carlsbad, CA, USA). Real-time monitoring of PCR was performed using the LightCycler System (Roche Applied Science, Indianapolis, IN, USA) and SYBR-Green I dye (Roche Diagnostics) as described previously [68 (link)]. Primer sequences and PCR conditions are listed in Table S2, Supplementary Information. The relative expression levels of these genes were obtained by normalizing mRNA expression to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression as an endogenous control in each sample.
RNAi-mediated knockdown of Nedd4L was obtained with Assay ID 136860 (ThermoFisher; Milan, Italy) following manufacturer procedures.

Both femur and tibia (in connection and including articulations and growth plates) of 6 days old mice were haversted after death, cleaned of surrounding soft tissue and frozen in liquid nitrogen. Bone samples were crushed with a mixer mill (Sartorius,Göttingen, Germany) using steel balls. RNA was extracted from the bone powder with Tri-Reagent (Sigma), then purified on columns according to the manufacturer's instructions (RNeasy Plus Mini Kit, Qiagen, Hilden, Germany). RNA amounts were assessed with the Ribogreen kit (Invitrogen, Life Technologies, Eugene, OR, USA) and their quality checked with the Experion automated electrophoresis station (BIO-RAD, Hercules, CA, USA). Messenger RNA was reverse-transcribed (iScript cDNA synthesis Kit, Biorad) according to manufacturer's instruction, then 400 ng of cDNA were amplified through QRT-PCR using the SYBR Green I dye (Lightcycler faststart DNA master SYBR green I, Roche, Germany). Expression of the genes of interest was normalized to glyceraldehyde-3-phosphate dehydrogenase (GADPH). The expression of the housekeeping gene did not differ at either age in either genotype. Primer sequences of the transcripts amplified are listed in Table 1.

Total RNA was extracted from HCT116 and HT29 cells using a RNeasy Plus Mini Kit (Qiagen, Venlo, Netherlands). Reverse transcription was performed using the SuperScript VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific). Polymerase chain reaction amplification was performed using the following primers: CDKN1A forward primer (5′-CCGGCTGATCTT CTCCAAGAG-3′) and reverse primer (5′-AAGATGTAGAGCGGGCCTTTG-3′), TP53 forward primer (5′-ATCCTCACCATCATCACACTGG-3′) and reverse primer (5′-ACAAACACGCACCTCAAAGC-3′), and TBP forward primer (5′-TGCTGCGGT AATCATGAGGATA-3′) and reverse primer (5′-TGAAGTCCAAGAACTTAGCTG GAA-3′) (Thermo Fisher Scientific). Real-time PCR was performed using a Light Cycler LC480 (Roche Diagnostics, Rotkreuz, Switzerland) with SYBR Green I dye (Roche) and TB Green Premix Ex Taq II (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. A constant amount of RNA was reverse transcribed to complementary DNA, and the complementary DNA was then amplified using the following conditions: 1 cycle of denaturation at 95 °C for 5 min, and 48 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 5 s, and extension at 72 °C for 3 s. The expression of each gene was normalized to TBP mRNA level and is represented as fold changes compared to the control group.