Superscript vilo cdna synthesis

For real-time analyses, RNA was extracted using RNeasy Plus Micro or Mini Kit (Qiagen) and complementary DNA obtained using SuperScript Vilo cDNA Synthesis (Invitrogen). When less than 10,000 cells were sorted, pre-amplification using TaqMan PreAmp (Applied Biosystems) was performed. Real-time reactions were assembled using Taqman probes (Applied Biosystems) in accordance with the manufacturer’s directions. Expression data were normalized by the expression of housekeeping genes ActB and Gapdh. A list of probes used for this study can be found in Table 1.Table 1

List of Taqman assays (mouse and human-specific) used for this study. Probes were purchased from Applied Biosystem.

Gene	Probe (human)	Probe (mouse)
Actin	Hs99999903_m1	Mm_00607939
GAPDH	Hs99999905_m1	Mm_99999915
Axin2	Hs00610344_m1	Mm00443610_m1
P16Ink4a	Hs00923894_m1	Mm_01257348
Bmi1	Hs00995519_g1	Mm03053308_g1
Usp16	Hs01062190_m1	Mm_00470393
Rspo1	Hs00543475_m1	Mm00507077_m1
Rspo2	Hs00379983_m1	Mm00555788_m1
Rspo3	Hs00262176_m1	Mm00661105_m1
Lgr5	Hs00969421_m1	Mm00438890_m1

Total RNA was extracted using the TRIzol (Invitrogen, Waltham, MA, USA) procedure from treated and untreated cell samples and the amount of RNA was determined with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher, Waltham, MA, USA). SuperScript Vilo cDNA synthesis (Invitrogen, USA) was used to create cDNA as follows: 1 μg of total RNA, 4 μL of the 5× Vilo reaction mix, 2 μL of the 10× SuperScript mix and RNA-free water were added to make a total volume of 20 μL. We used β-actin as an internal control and a semiquantitative PCR was used to measure the expression of Bax, Bcl-2 and caspase-7 in MCF-7 cells. The final volume (20 μL) of the RT-PCR mixture contained 4 μL of 5X FIRE pol Master Mix, 10 pmol of each specified primer and double-distilled water. The sequences of the primers used were as follows: Bax, F: 5′-CGGGTTGTCGCCCTTTTCTA-3′, R: 5′-AAAGTAGGAGAGGAGGCCGT-3′; Bcl-2, F: 5′-TGATGCCTTCTGTGAAGCAC-3′, R: 5′-ACAGGCGGAGCTTCTTGTAA-3′; caspase-7, F: 5′-AGTGACAGGTATGGGCGTTC-3′, R: 5′-TCCATGGCTTAAGAGGATGC-3′; β-actin; F: 5′-ACTGGGACGACATGGAGAAAA-3′, R: 5′-GAGGCGTACAGGGATAGCAC-3′. The 20-µL final amplification products were run on 1.2% agarose gel stained with ethidium bromide and a gel image was captured with LICOR gel doc.

Experiment was performed in triplicate, and a total of 9 scaffolds of each group were used. Cells were collected 3, 7, and 14 days after plating. Media were removed, and scaffolds were rinsed twice with cold phosphate-buffered saline. For evaluation of mRNA expression on the titanium alloy scaffolds, adherent cells in each sample were lysed using TRIzol (Invitrogen, Carlsbad, CA). Cell lysates were collected by pipetting and centrifugation. Samples were kept frozen at 80°C for at least 24 hours. Total RNA in the cell lysates was collected by ethanol precipitation, according to the manufacturer's protocol. Total RNA was quantified using a spectrophotometer (PowerWave HT-BioTek Instruments) and the Gen5™ program (BioTek Instruments). From each total RNA sample, cDNA was generated using SuperScript VILO cDNA Synthesis (Invitrogen) in a standard 20 µl reaction using 50 ng of the total RNA. Subsequently, equal volumes of cDNA were used to program qPCR reactions specific for mRNAs encoding the early osteogenic marker Runx2 (Qiagen, Germantown, MD). The qRT-PCR reaction was performed in an Applied Biosystems 7900HT Real Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA abundance was determined by the 2^−ΔΔCt method and reported as a fold change. TiAlV scaffold at day 3 was used as control group. GAPDH abundance was used for normalization.

Total RNA from cells was extracted using a High Pure RNA Isolation kit (Roche, Basel, Switzerland). cDNA was obtained from 1 μg of RNA by SuperScript VILO cDNA synthesis (Invitrogen) in accordance with the manufacturer’s instructions. ChIP-qPCR was performed using the SYBR Green PCR Master Mix and an ABI Prism^® 7900HT instrument (Applied Biosystems^®, Waltham, MA, USA). Primers were designed using OligoPerfect Designer™ (Invitrogen), and reactions were performed in triplicate. The relative amount of each amplified fragment was estimated with respect to the amplification obtained from input DNA and corrected by GADPH expression via the 2^−ΔCt method. The primer sequences used for qPCR assessment of the mRNA levels of target genes are listed in Table S1.