Moloney murine leukemia virus m mlv reverse transcriptase

Total RNA was prepared using ISOGENII (Nippon Gene). One microgram of total RNA was synthesized into cDNA using random hexamer primers (Takara Bio) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Thermo Fisher Scientific.). RT‒qPCR was performed using TB Green Premix Ex Taq II (Takara Bio) and a Thermal Cycler Dice Real Time System II (Takara Bio). The PCR conditions were 95 °C for 30 s, 30 cycles of 95 °C for 5 s, 60 °C for 30 s and 95 °C for 15 s, and 60 °C for 30 s and 95 °C for 15 s. All target gene expression levels were normalized to β-actin or GAPDH expression. The sequences of the primers used in this study are listed in Table S4.

Total RNA was extracted from the spleen and tumor tissues using a total RNA isolation kit (DENAzist Asia, Mashhad, Iran). The extracted total RNA was subjected to 1U of DNase I (RNase free) (Thermo Scientific Fisher, Waltham, MA, USA) at 37°C for 15 min. The inactivation of the DNase was conducted by adding 1 μL of 50 mM ethylenediaminetetraacetic acid (EDTA) (Merck). Moloney murine leukemia virus (M-MLV) reverse transcriptase (Thermo Fisher Scientific) was used to convert RNA samples to cDNA. The quantitative real-time polymerase chain reactions (qRT-PCR) were carried out using RealQ Plus 2x Master Mix Green (Ampliqon A/S, Odense, Denmark) in a Rotor-Gene^® Q real-time PCR cycler (QIAGEN, Hilden, Germany). The amplification steps were as follows: 15 min at 95°C (1 cycle), followed by 30 s at 95°C, 30 s at 58°C–62°C, and 25 s at 72°C (35–40 cycles). Three replicates were considered for qRT-PCR readout. The β-actin gene served as the internal control. Table 2 gene-specific primers and their accession numbers used in this study.

Total RNA was prepared using ISOGENII (Nippon Gene, Tokyo, Japan). One microgram of total RNA was utilized for cDNA synthesis using random hexamer primers (Takara Bio, Ohtsu, Japan) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Thermo Fisher Scientific Inc. Rockford, IL, USA). qRT-PCR was conducted using SYBR Premix Ex Taq II (Takara Bio, Ohtsu, Japan) and a Thermal Cycler Dice Real Time System II (Takara Bio, Ohtsu, Japan). The PCR conditions were 95 °C for 30 sec, 30 cycles of 95 °C for 5 sec, 60 °C for 30 sec and 95 °C for 15 sec, 60 °C for 30 sec and 95 °C for 15 sec. All target gene expression levels were normalized to β-actin or Gapdh expression. The sequences of the primers used are listed in Table S2.

The cells (2.0 × 10⁴ cells/well) were cultured on non-tissue culture-treated 12-well plates with the growth medium for five days. Then, BA (+) (0.1%, 0.5%, 1%, and 5% (v/v)), 0.5% BA (−) (v/v), and 0.5% SC (v/v) were added to the mineralization medium. The total RNA was extracted using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA) on day 7. Moloney murine leukemia virus (M-MLV) reverse transcriptase (Thermo Fisher Scientific) was used to reverse transcribe RNA (1 µg) to complementary DNA in a 20 µL reaction system (cDNA: 1 µL; forward primer: 1 µL; backward primer: 1 µL; FastStart Essential DNA Green Master PCR grade H₂O (Roche, Basel, Switzerland): 7 µL; FastStart Essential DNA Green Master 2× conc. (Roche): 10 µL) according to the method previously reported [29 (link)]. The mRNA expressions of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) as dentinogenesis-related genes were quantified using quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR). Quantitative real-time RT-PCR was performed using a LightCycler^® Nano (Roche) according to the manufacturer’s instructions. Table 2 presents the primer sequences and PCR conditions for the LightCycler^® Nano. The comparative 2^−ΔΔCT method was applied to determine the relative changes in the gene expression compared to the reference housekeeping gene β-actin.

For RNA isolation, R. favelukesii LPU83 was cultivated in TY media. Then, RNAprotect (Qiagen, Hilden, Germany) reagent was added, the cells were harvested, and the pellet was frozen in liquid nitrogen. The commercial RNeasy R Protect Bacteria Mini Kit (Qiagen, Hilden, Germany) was used for the RNA extraction, and the DNA was removed with DNase I (Qiagen, Hilden, Germany).
The total RNA was retrotranscribed to cDNA using a Moloney murine leukemia virus (MMLV) reverse transcriptase (Thermo Fisher Scientific, Buenos Aires, Argentina), using random hexamers as primers.