Superscript 3 rnase h reverse transcriptase kit

Total RNA samples were extracted from liver and gill tissues using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. The purity and concentration of RNA were detected with Nanodrop 2000 spectrophotometer (Thermo scientific, USA), accompanied with the integrity of RNA checked by 1.0% agarose gel electrophoresis. One microgram of the resulting total RNA was reverse transcribed into cDNA using the SuperScript III RNaseH-Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA) and oligo dT primers (Promega, Charbonnie `res, France) according to the manufacturers’ instructions.
Quantitative RT-PCR was carried out on an iCycler iQTM real-time PCR detection system (BIO-RAD, Hercules, CA, USA) using the Eva Green 2 × qPCR Master mix (ABM, Canada). The program was as follows: 95 °C for 5min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. In addition, a melt curve analysis was performed after amplification to verify the accuracy of each amplicon. 18S was employed as a non-regulated reference gene and no changes in 18S gene expression were observed in our investigations (data not shown). The relative quantification of the target genes was determined via the comparative CT method (2^-ΔΔCt method). Primer used in the present study was shown in Table 2.

Relative hepatic gene expression was determined by quantitative real-time RT-PCR. The extraction of total RNA was performed using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. An amount of 1 μg of total RNA was used for cDNA synthesis. The NCode™ VILO™ miRNA cDNA synthesis kit (Invitrogen), or the SuperScript III RNAse H– Reverse transcriptase kit (Invitrogen) with random primers (Promega, Charbonniéres, France), were used to synthesize cDNA (n = 6 for each treatment) for miRNA and mRNA, respectively.

Total RNA was extracted from leaves using Trizol reagent according to the manufacturer’s instructions (Tiangen, China). The quality of the extracted RNA was examined by agarose gel electrophoresis. The first strand cDNA was synthesized using the Superscript™ III RNase H-Reverse Transcriptase kit (Invitrogen, USA). The full-length cDNA sequence of CsLEA11 was amplified using gene-specific primers designed according to the cucumber genome initiative (CuGI, http://cucumber.genomics.org.cn, Gene ID: Csa020767), and NCBI database (GenBank Accession Number: XM_004150027.2). The PCR product was cloned into the pGEM-T (Promega, USA) and subsequently sequenced.
The theoretical pI, MW and the grand average of hydropathy index (GRAVY) of the deduced CsLEA11 protein were predicted by the ProtParam program (http://web.expasy.org/protparam). The prediction of protein secondary structure and intrinsic disorder in the deduced CsLEA11 protein was performed by the PHYRE2 protein fold recognition server (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index). The multiple sequence alignments of CsLEA11 and other dehydrin proteins were performed with Clustal W software (Larkin et al. 2007 (link)). A phylogenetic tree was then constructed with the MEGA 5.0 software by the neighbor-joining method with 1000 bootstrap replicates (Tamura et al. 2007 (link)).

Using 400 μM AlCl₃ solution to irrigate (each pot soil weighing 2 kg) and when the artificial climate indoor pot (at 28°C, 16 h light/ 8 h dark) was cultured for 1 year, Zoysia japonica varieties "Company" was grown up to 20 cm. Twenty plants per pot were planted and similarly three pots were repeated. The plants were treated for 10 consecutive days, and the roots and leaves of each basin were collected, respectively. Total RNA was extracted from leaf and root of Zoysiagrass using TRIzol reagent according to the manufacturer’s instructions (Tiangen, China). The first strand cDNA was synthesized using the Superscript™ III RNase H-Reverse Transcriptase kit (Invitrogen, USA).

An adult female centipede S. dehaani Brandt, 1840 (Scolopendromorpha, Chilopoda) was obtained from a local pet shop. Selected tissues of S. dehaani were dissected, shock-frozen in liquid N₂ and kept at − 80 °C until use. Total RNA was extracted either according to the method by Holmes and Bonner [44 (link)] or with the RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. The quality of the RNA was checked by measuring the OD 260/280 ratio and by gel electrophoresis. cDNA was obtained using the SuperScriptTM III RNase H-Reverse Transcriptase Kit (Invitrogen, Karlsruhe, Germany), employing an oligo(dT)-primer. Partial S. dehaani Hc and PO sequences were obtained with PCR using sets of primers that had been generated on the basis of partial cDNAs [35 (link)] (Additional file 1: Table S1). Missing 5′ and 3′ ends were completed by the RACE technique using the GeneRacer™ kit (Invitrogen) according to the manufacturer’s instructions. The PCR products were cloned into the pGEM-T vector (Promega, Mannheim, Germany) and sequenced by a commercial service (GATC, Konstanz, Germany).

Reverse transcription was performed with 775 ng total RNA from eggs and fat body of S. dehaani employing the SuperScriptTM III RNase H-Reverse Transcriptase Kit (Invitrogen) and oligo(dT)20 primer according to the manufacturer’s instructions. Quantitative real-time reverse transcription PCR (qRT-PCR) was performed with primer sets specific for Hc subunits, PO, β-actin and RPLP0 (Additional file 1: Table S1) using the Power SYBR Green PCR Master Mix and the 7500 Real-Time PCR System (Applied Biosystems, Darmstadt). qRT-PCR reactions were performed in technical triplicates. Amplification was carried out using a standard PCR protocol (95 °C for 15 s, 58 °C for 15 s, and 72 °C for 30 s; 40 cycles) and fluorescence was measured at the last step of each cycle. The specificity of the amplification reactions was validated by analysis of the dissociation curve. The mRNA copy numbers were calculated with the standard curve approach, which employs a dilution series with plasmids carrying the respective cDNA sequences [53 (link)]. Calculations were performed with the 7500 Software 2.0.6 (Applied Biosystems).