Revert aid first strand complementary dna synthesis kit

For analysis of mRNA levels, total RNA from the 1-mm layer of femur tissues around implants were prepared and isolated using TRIzol reagent (Invitrogen). Complementary DNA was synthesised using the RevertAid First Strand Complementary DNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA). Quantitative real-time PCR was performed using the SYBR Premix Ex TaqII (Takara, Dalian, China), as per the manufacturer’s instructions. U6 and GAPDH were used as internal controls. Relative expression was calculated using the 2^−ddCt method. The primer sequences used are shown in Additional file 1: Table S2.

Total RNA was isolated using the Nucleospin RNA kit or RNA XS kit (both from Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. Complementary DNA was synthesized from 0.25 to 1.0 μg RNA using the RevertAid First Strand Complementary DNA Synthesis Kit (Thermo Fisher). Real-time qPCR was performed with SYBR Green Master mix (Bio-Rad, Hercules, CA) using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Expression values were normalized to β-actin expression. The sequences of all primers used can be found in Table 1.Table 1

All Primer Sequences

Gene	Forward primer, 5’---3’	Reverse primer, 5’---3’
CTSH	TACCTTCGAGGTACTGGTCCCT	GGTGGAGAAAGTCCAGCAACTG
WNT2	AGGATGCCAGAGCCCTGATGAA	AGCCAGCATGTCCTGAGAGTAC
PI16	CTGGTGTGCAACTATGAGCCTC	GGCAAATCCTGAGCATCTTCCG
SCUBE3	CTCCAGGCAAAGAGGTCACAAG	TCCTTTCAGCCGCCGTTCCATT
SEMA3C	ACCCACTGACTCAATGCAGAGG	CAGCCACTTGATAGATGCCTGC
ACTA2	CCGGGAGAAAATGACTCAAA	GAAGGAATAGCCACGCTCAG
VIM	TGTCCAAATCGATGTGGATGTTTC	TTGTACCATTCTTCTGCCTCCTG
FAP	ATCTATGACCTTAGCAATGGAGAATTTGT	GTTTTGATAGACATATGCTAATTTACTCCCAAC
CD31	GCTGACCCTTCTGCTCTGTT	TGAGAGGTGGTGCTGACATC
CD45	AACAGTGGAGAAAGGACACA	TGTGTCCAGAAAGGCAAAGC
KRT20	CAGACACACGGTGAACTATGG	GATCAGCTTCCACTGTTAGACG
ACTB	GTTGTCGACGACGAGCG	GCACAGAGCCTCGCCTT

Total RNAs from cells were isolated using TRIzol reagent (Invitrogen). Subsequently, 2 μg RNA was reverse transcribed into synthesize complementary DNA in a 20 μl reaction mixture using RevertAid First Strand complementary DNA Synthesis Kit (K1622, Thermo Fisher Scientific). Real-time reverse-transcription PCR was carried out by SYBR premix Ex TaqII Kit (Takara). Actin was performed in parallel as a control. The mRNA expression of each gene was quantified by measuring cycle threshold values. The relative expressions were calculated using the 2^−ΔΔCt method. The primers for RT-qPCR are shown in Table S2.

Total cellular RNA was extracted using TRIzol (Invitrogen, USA) and random primed RNAs (1 μg) were reverse transcribed with RevertAid first-strand complementary DNA synthesis kit according to the manufacturer’s instructions (Thermo Scientific, USA). Relative expression of the mRNA was calculated by 2^−ΔΔCt method and normalized to β-ACTIN. Real-time PCR was performed using ABI QuantStudio (Applied Biosystems, CA, USA). Values are means ± SD from three independent experiments which were performed in duplicate. Statistical comparisons among groups were performed using a One-way ANOVA followed by Boferroni’s test. Specific PCR primer sequences are listed in Supplementary Table 2.

Total RNA of samples was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA (~1.5 μg) from each sample was reverse transcribed into cDNA using a Revert Aid First Strand Complementary DNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RT-qPCR was performed using an RT-qPCR kit (Novoprotein Scientific, Inc.) on a QuantStudio™ 6 Flex Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). GAPDH was used as a reference. The primer pairs used for qPCR in the study is listed in Table S2. The relative expression of each target was analyzed using the 2^‑ΔΔCt method 40 (link).

The cells were cultured in 6-well plates with leaching liquids at 4 × 10⁴ cells/well. After 7 days of co-culture, total RNA was extracted using a 1 mL/well TRIzol RNA Extraction Kit (Invitrogen, Carlsbad, CA, USA). Next, the RNA was reverse transcribed into complementary DNA using the Revert Aid First Strand Complementary DNA Synthesis Kit (Thermo Fisher Scientific, San Jose, CA, USA). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to examine the gene expression levels of OCN, OPN, Col-1, Runx-2, VEGF, and HIF-1α; human β-actin was set as a control. The sequences are designed from NCBI and synthesized by Servicebio, which are listed in Supplementary Table S1.