Anchored oligo dt 18 primer

Total RNA was extracted with High Pure RNA Isolation Kit (Roche), according to the manufacturer’s instructions. RNA was quantified in a NanoDrop 2000c (Thermo Scientific) and used for cDNA synthesis. Reverse transcription was performed with High Fidelity cDNA Synthesis Kit (Roche), using Anchored-oligo(dT)18 Primer (Roche) or with the Super Script III First Strand synthesis system (Invitrogen), using random hexamers (Invitrogen). qPCRs were performed in triplicates using LightCycler 480 SYBR Green I Master Kit (Roche) with the following primers: βIII-tubulin (TUBB3; fwd 5′-GGGCCTTTGGACATCTCTTC-3′ and rev 5′-CCTCCGTGTAGTGACCCTTG-3′), glial fibrillary acidic protein (GFAP; fwd 5′-AGAGAGGTCAAGCCCAGGAG-3′ and rev 5′-GGTCACCCACAACCCCTACT-3′) and ribosomal protein L22 (RPL22; fwd 5′-CACGAAGGAGGAGTGACTGG-3′ and rev 5′-TGTGGCACACCACTGACATT-3′). The reactions were performed with LightCycler 480 Instrument II 96-well block (Roche). Quantification cycle values (C_q’s) and melting curves were determined using LightCycler 480 Software version 1.5 (Roche). All data were analyzed using the 2^−ΔΔCt method for relative gene expression analysis47 (link). Changes in gene expression were normalized using the housekeeping gene RPL22 as internal control.

Cells exposed to the tested fluoroquinolones at concentrations of LC₁₀, LC₅₀, and LC₉₀ were analyzed for the expression of selected genes (BAX, BCL2, TOP2A, TOP2B, and CDKN1A). Total RNA from tested cells was purified using a RNeasy Mini Kit (Qiagen, Germany). The cell pellet was disrupted with an appropriate volume of lysis buffer and processed according to the manufacturer’s protocol. The obtained material was subjected to qualitative and quantitative analysis using the NanoDrop™ Lite Spectrophotometer (Thermo Fisher Scientific, USA). Reverse transcription reactions for complementary DNA synthesis were performed using the Transcriptor First Strand cDNA Synthesis Kit with the Anchored-oligo(dT)₁₈ Primer (Roche, Switzerland) according to the manufacturer’s recommendations. Gene expression was evaluated by quantitative polymerase chain reaction (qPCR) using LightCycler^® 480 SYBR Green I Master (Roche, Switzerland) and PCR primers (Bio-Rad, USA) for selected genes with SDHA and TBP as the reference genes. The obtained gene expression results were compared to control cells and subjected to statistical analysis.

Total RNA was directly converted to cDNA using the M-MLV Reverse Transcriptase (RT, Invitrogen, Waltham, MA, USA). cDNA synthesis was performed with approximately 500 ng of RNA in a final volume of 20 µL containing 20 U/µL of RT, 2.5 µM of anchored-oligo(dT)₁₈ primer (Roche, Basel, Switzerland), 60 µM of random hexamer primer (Roche), 20 U of RNase inhibitor (Promega, Walldorf, Germany), and 1 mM each deoxynucleoside triphosphate (dNTPs, Promega, Walldorf, Germany).
According to the manufacturer’s protocol, secondary structures were denatured by heating samples for 10 min at 65 °C in the presence of anchored-oligo(dT)₁₈ primer, random hexamer primer, dNTP mix, and water PCR grade in a final volume of 20 µL. Samples were then cooled on ice immediately, then supplemented with 5X First-Strand Buffer, 0.1 M dithiotrethol (DTT) and 40U of RNaseOUT™ Recombinant Ribonuclease Inhibitor, and incubated at 37 °C for 5 min. One microliter (200 U) of M-MLV RT was added and each sample was incubated at 25 °C for 10 min followed by 50 min at 37 °C. Finally, the enzyme was inactivated by heating at 70 °C for 15 min. Two negative controls were performed in each reaction: one without RNA and the other without enzyme (RT minus control allowing the detection of eventual genomic DNA contamination).

RNA was reverse-transcribed using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (RT, Invitrogen). Complementary DNA (cDNA) synthesis was performed with ~500 ng of RNA in a final volume of 20 μl containing 20 U/μl of RT, 2.5 μM of anchored-oligo(dT)₁₈ primer (Roche), 60 μM of random hexamer primer (Roche), 20 U of RNase inhibitor (Promega), and 1 mM each dNTP.
According to the manufacturer's instructions, secondary structures were denatured by heating samples for 10 min at 65°C in the presence of anchored-oligo(dT)₁₈ primer, random hexamer primer, dNTP mix, and water PCR grade in a final volume of 20 μl. Samples were then cooled on ice immediately, briefly centrifuged, supplemented with 5X First-Strand Buffer, 0.1 M dithiothreitol (DTT) and 40 U of RNaseOUT™ Recombinant Ribonuclease Inhibitor, and incubated at 37°C for 5 min. One microliter (200 U) of M-MLV RT was then added, and each sample was incubated at 25°C for 10 min followed by 50 min at 37°C. The enzyme was inactivated by heating at 70°C for 15 min. Two negative controls were performed in each reaction, one without RNA and the second without the RT enzyme (RT minus control allowing the detection of eventual genomic DNA contamination).

For this experiment, 10⁶ of MSCs were seeded in 6-well plates and cultured in complete Mesenchymal Stem Cell Medium Kit for 24 h. Then, the medium was replaced, and the cells were treated with PA and its derivatives solutions at 10 µg/mL in complete LG-DMEM. Media containing the corresponding compounds were refreshed every 2 d. Total RNA was prepared using the RNeasyMini Kit (Qiagen), following the manufacturer’s instructions. To quantify the levels of ALPL and COL1A1 mRNA, complementary DNA was prepared from total RNA using the Transcriptor Reverse Transcriptase and an anchored-oligo (dT)18 primer (Roche Applied Science). qPCR was performed using LightCycler FastStart DNA Master SYBR Green I and LightCycler detector (both from Roche Applied Science). Quantitative expression values were extrapolated from standard curves, and were normalized to the expression values of beta-2-microglobulin (B2M) and beta-glucuronidase (GUSB) which were used as endogenous controls. Specific oligonucleotide primers were: COL1A1, 5′-CGGGCCTCAAGGTATTGCT-3′ (forward primer, F) and 5′-GGGACCTTGTTTGCCAGGTT-3′ (reverse primer, R); ALPL, 5′-GACTAAGAAGCCCTTCACTGCCAT-3′ (F), 5′-GACTGCGCCTGGTAGTTGTT-3′ (R); B2M, 5′-CCAGCAGAGAATGGAAAGTC-3′ (F) and 5′-GATGCTGCTTACATGTCTCG-3′ (R); GUSB, 5′-AAACGATTGCAGGGTTTCAC -3′ (F), 5′-CTCTCGTCGGTGACTGTTCA-3′(R).