Ribonuclease inhibitor

Ribosome in complex with Fe(II) tethered ArfA was probed by initiating hydroxyl radical formation with 6 μl of 250 mM ascorbic acid and 6 μl of 1.25% H₂O₂. The reaction mixtures were incubated on ice for 10 min and quenched with 100 mM thiourea. RNA was prepared by phenol extraction and ethanol precipitation. reverse transcriptase reaction was carried out in a 12 μl reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM each of dNTP, 1 pmol of rRNA, 1 pmol of 5′Texas Red labeled DNA primer complementary to a portion of the rRNA sequence, three units of ribonuclease inhibitor (Takara) and 18 units of reverse transcriptase (Takara). After the addition of 3 μl of stop solution containing 95% formamide, 0.5 mM EDTA and 0.1 mg/ml Fuchsin Red, the positions of cleavages were analyzed using a fluorescence DNA sequencer (Hitachi SQ-5500).

The mRNA concentrations of spleens for broiler chickens TNFα, IL6, IFNγ, NF-κB p65, and HO-1 were quantified by quantitative real time PCR. β-actin was used as a house-keeping gene to normalize the gene expression data. The primer information for all the genes is listed in Table 1.
Total RNAs were extracted from the spleens by TRIZOL Reagent Kit (Invitrogen, San Diego, CA, USA). Reverse transcription (RT) was carried out using an RT reactions (10 μL) consisted of 500 ng total RNA, 5 mmol/L MgCl₂, 1 μL RT buffer, 1 mmol/L dNTP, 2.5 U AMV, 0.7 nmol/L oligo d(T) and 10 U ribonuclease inhibitor (TaKaRa, Dalian, China). The cDNA was amplified in a 20 μL PCR reaction containing 0.2 μmol/L of each specific primer (Sangon, Shanghai, China) and SYBR green master mix (TaKaRa, Dalian, China). Each cycle consisted of denaturation at 95 °C for 10 s, annealing at 95 °C for 5 s, and extension at 60 °C for 34 s. Each sample was measured in duplicate analysis. If the difference between two duplications was greater than 15%, the sample was analyzed again. The PCR products were verified by electrophoresis on a 0.8% agarose-gel and by DNA sequencing. Standard curves were generated using pooled cDNA from the samples being assayed, and the comparative cycle threshold (C_t) method (2^−ΔΔCt) was used to quantitate mRNA expression according to Livak and Schmittgen [55 (link)].

Reverse transcription (RT) was carried out in 20 μl solution that contained 0.2 μl 200 U/μl MMLV reverse transcriptase (PROMEGA), 0.2 μl 40 U/μl ribonuclease inhibitor (TAKARA), 0.8 μl 10 mmol/ml dNTP mix (TAKARA), 1.2 μl 10 mmol/ml stem-loop RT primer (SANGON), 4 μl MMLV RT buffer (PROMEGA), 11.6 μl RNAase-free water (PROMEGA), and 2 μl microRNA template. After being mixed gently, the reaction mixtures were incubated at 25 °C for 5 min, 40 °C for 60 min and then 70 °C for 15 min. The final cDNA products were stored at −20 °C until use. The reverse transcription primers are miR-24 [GenBank: AF480527.1], 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGATACGACCTGTTCCT-3′; miR-320a [GenBa-nk: JA682606.1], 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTCGCCCTC-3′; miR-423-5p [GenBank: JA830813.1], 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAAAGTCTC-3′; cel-miR-39 [GenBank: AJ487564.1], 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCAAGCTGA-3′.

cDNA was prepared by reverse transcription using total RNA as the template, and 1 μg of each RNA sample heat-treated at 65 °C was mixed with 20% five First Strand Buffer (Thermo Fisher Scientific), 1 mM dithiothreitol (Life Technologies, Gaithersburg, MD, USA), and 1 μg of each RNA sample heat-treated at 65 °C. The final concentration shown below was added to the sample: (Gaithersburg, MD, USA), 1.1-U/µL ribonuclease inhibitor (Takara Bio, Shiga, Japan), 0.5-mM dNTP mixture (Takara Bio), 5-U/µL Moloney-Mouse leukemia virus reverse transcriptase (Life technologies), and 55 ng/µL random Hexamers (Pharmacia Biotech, Milwaukee, WI, USA). The reaction solution was stored at 37 °C for 60 min and subsequently treated at 99 °C for 5 min to inactivate the residual enzymes for cDNA preparation.

The reverse transcription (RT) was carried out in a 20 μL reaction mixture containing 40 U/μL Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, WI, USA), 20 U/μL ribonuclease inhibitor (TAKARA, Dalian, China), 40 nmol dNTP mix (TAKARA, Dalian, China), 10 μmol stem loop RT primer (SANGON, Shanghai, China), 2 μL template miRNA, 4 μL MMLV RT buffer (Promega, WI, USA) and 11.6 μL RNAase-free water (Promega, WI, USA). After being mixed gently, the reaction mixtures were incubated at 25 °C for 5 min, 42 °C for 60 min and then 70 °C for 15 min. The final cDNA products were stored at − 30 °C until use. Each sample was repeated twice and RNase-Free water, instead of template miRNA, was used as negative control in RT.

Total RNA was isolated from nematodes using Trizol (Invitrogen, UK) according to manufacturer’s protocols. RNAs were first assessed for their purity and concentration by OD260/280 in a spectrophotometer, and then used for cDNA synthesis performed in a 12.5 μL reaction volume containing 625 ng total RNA, 0.5 mM reverse-transcript primers, 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 20 units ribonuclease inhibitor and 100 U reverse transcriptase (Takara, China). After cDNA synthesis, relative expression levels of examined genes were determined by real-time PCR in an ABI 7500 real-time PCR system with Evagreen (Biotium, USA). Relative quantification of targeted genes in comparison to reference tba-1 gene encoding tubulin was determined. The final results were expressed as relative expression ratio between the targeted gene and the reference gene. Primer information was shown in Table S7. All reactions were performed in triplicate.

Total RNA was isolated from ESCC cells with Trizol reagent (Invitrogen) according to the manufacturer's protocols. NanoDrop 2000 (Thermo Scientific) was used to detect the purity and quality. Reverse transcribed 2 μg RNA using reverse transcriptase M‐MLV (Takara Bio Inc.), ribonuclease inhibitor (Takara Bio Inc.) and dNTP mixture (Takara Bio Inc.). The RT products were 1:5 diluted and subjected to quantitative polymerase chain reaction (PCR) using a SYBR Green PCR Kit (Takara Bio Inc.) on a Roche 480 Real‐Time PCR System (Roche). GAPDH was applied for the endogenous reference gene. The expression level of Mig‐6 mRNA was presented as 2^{‐averageΔΔCT} × 100%. The primers sequences used are listed in Table 1. The experiment was performed in triplicate.