Superscript r 2 reverse transcriptase

Total RNA was extracted from cells, micromass, and HA composite scaffolds using Trizol reagent (Life Technologies) and cDNA was synthesized by using Superscript(R) II reverse transcriptase following the manufacturer’s protocol (Invitrogen, Carlsbad, CA). Real-time PCRs were performed using StepOnePlus(R) Real Time PCR System (Applied Biosystems, Carlsbad, CA) with SYBR Green PCR Master Mix (Life Technologies). The relative expression of each target was calculated using the ΔΔCT method and β-actin were used as endogenous references. All expression levels of samples were normalized to controls. The PCR primers used for RT-PCR are listed in Table S1. Expression of all markers was normalised to the expression of the housekeeping gene β-actin.

We followed the MIQE guidelines (Bustin et al. 2009 (link)) for RT-qPCR validation of DEGs identified from transcriptome analysis. Primer Express R software (Applied Biosystems) and OligoAnalyzer 3.1-IDT were used to design and evaluate all specific primers. The total bacterial RNA was extracted by using the Trizol reagent (Ambion, Texas, USA), following the manufacturer’s recommendations and treated with 5U of Amplification Grade DNase I (Invitrogen, Carlsbad, CA, USA) for 10 min at 70 °C. After DNase I denaturation, the RNA sample was chilled on ice and immediately used with Oligo(dT) 20 primers (Invitrogen, Carlsbad, CA, USA) and SuperScript R II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) to synthesize cDNA. For RT-qPCR analysis, the amplification was performed with a Step One Plus Real-Time PCR System (Applied Biosystems). The reaction was performed under the following conditions: 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 40 s. Amplicons were identified by melting curve analysis and the 16 s rRNA gene was employed as a stable reference gene after cycle completion. Three biological and three technical replicates were performed for each sample. Gene expression levels were estimated using the 2−^ΔΔCT method (Livak and Schmittgen 2001 (link)).