Random hexamer primer

Ten medaka or zebrafish embryos for each stage were used to collect total RNA with Sepazol (SIGMA). Total RNAs were treated with RQ1 DNase to prevent genomic DNA contamination. One μg of total RNA for each stage and species was used for synthesis of first-strand cDNA with the M-MLV Reverse Transcriptase (Promega) using Random hexamer primer (Promega). The resultant cDNA samples were subjected to semi-quantitative PCR amplification (30 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 1 min) and run in an agarose gel. The band of PCR products was evaluated in comparison with a housekeeping gene, ef1α. Primer information is listed in Supplementary S1 Table.

Cells were harvested from cultures cultivated for 4, 7, 12, 16, 18, and 24 h by centrifugation at 3,300 g at 4°C for 6 min, pellets were homogenized with 1 ml of lysis solution Extract-All (Eurobio) according to manufacturer's guidelines. After that, 200 μl of chloroform were added to the suspension allowing separation of cell components. Water phase containing RNA was removed, precipitated with 500 μl of isopropanol and dissolved again in 50 μl of RNase-free water after washing in 75% cool ethanol. Samples were then treated with DNase I (Sigma-Aldrich) and purified using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Possible DNA contamination was detected by PCR detecting house-keeping gene flaA (Nachamkin et al., 1993 (link)). The integrity of RNA was verified using the Experion System (Bio-Rad). Its concentration and purity was measured using NanoDrop 2,000 (Thermo Scientific).
For reverse transcription 100 ng of RNA were mixed with 0.5 μl of 1 μM Random Hexamer Primer (Promega) and 4.5 μl of RNase-free water. This mixture was incubated 5 min at 70°C followed by 5 min on ice. After that 15 μl of Master Mix were added to mixture (5 μl of 5 × RT buffer, 1 μl 25 mM dNTP and 1 μl of M-MLV H⁻ reverse transcriptase; Promega) and it was incubated 10 min at room temperature, 50 min at 48°C and finally 15 min at 70°C to stop the reaction.

Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. The cDNA was synthesized from 1 μg of RNA using M-MuLV Reverse Transcriptase (NEB) and random hexamer primer (Promega). qPCRs were run on LightCycler® 480 Instrument II (Roche) using 2X SYBR green mix (Bio-rad). The primers used in the qPCR were as follows: mouse TNF-α sense 5′-GCCTCTTCTCATTCCTGCTTG-3′, antisense 5′-CTGATGAGAGGGAGGCCATT-3′; mouse Gapdh sense 5′-CATGGCCTTCCGTGTTCCT-3′, antisense 5′-TGATGTCATCATACTTGGCAGGTT-3′; Dusp1 sense 5′-GGCCAGCTGCTGCAGTTTGAG-3′, antisense 5′-AGGTGCCCCGGTCAAGGACA-3′.

For the first strand, cDNA was synthesized using reverse transcriptase (Fermentas, United States) and buffer (5X) [50 mM Tris-HCl (pH 8.3 at 25 OC), 250 mM KCl and 20 mM MgCl₂ and 50 mM DTT] in the presence of a random hexamer primer (Promega, United States) and 5 μl of RNA was added to [10 μl (5x) RT—buffer, 5 μl (25 mM) dNTPs, 5 μl of primer, 0.5 μl (20 U/ μl) of RT—enzyme, 24.5 μl H₂O]. The mixture was incubated at 37°C for 60 min and then at 70°C for 10 min (for enzyme inactivation) followed by storage at 4°C until use (Sambrook and Russell, 2001 ).

Plant total RNA was extracted from 2-week-old seedlings using Trizol Reagent (Invitrogen, CA, USA), and the first-stand cDNA was synthesized by total RNA with SuperScript II RNase H2 reverse transcriptase (Invitrogen) using random hexamer primer (Promega). Semi-quantitative RT-PCR was carried out using gene-specific primers. ACTIN8 was used as an internal control. RT-qPCR was performed according to the instructions provided for the Bio-Rad iCycler iQ system (Bio-Rad Laboratories, CA, USA) using platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). The fold change of transcripts was calculated based on an efficiency calibrated model [62 (link)] and each sample were quantified at least in triplicate and normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as an internal control. Statistical differences between the samples were evaluated by Student’s t-test or ANOVA in combination with post-hoc test using delta Ct values [62 (link)]. The specific primers used are listed in S1 Table.

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).