Mmlv rt reverse transcriptase

Total RNA was extracted from the larval samples and seven other tissues, including gill, adductor muscle, viscera, gonad, foot, mantle pallial, and mantle edge of adults using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and a standard procedure. RNA was quantified at optical densities of 260/280 with an Utrospec 3000 UV-visible spectrophotometer (Amersham Biosciences, Uppsala, Sweden). RNA integrity was determined by fractionation on a 1.2% formaldehyde denatured agarose gel stained with Goldview.
Gene expression profiling during larval development was accomplished by DNA microarray using pooled samples from different stages (fertilized eggs, trochophore stage, D-shaped stage, and umbonal stage larvae, as well as juveniles). RNA was extracted as described above. Triplicate pooled samples were run for each stage point.
First-strand cDNA was produced for real time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using 1 μg total RNA from pooled samples of the different larval stages using PrimeScript™ RT Master Mix (Perfect Real Time; Takara Bio, Shiga, Japan). Tissue-specific semi-quantitative PCR was conducted using 1 μg total RNA of the different adult tissues to synthesize first-strand cDNA using MMLV-RT reverse transcriptase (Promega, Madison, WI, USA).

Total RNA was extracted from mouse and human tissue and mouse-derived cultured cell lines using TRIzol reagent from the Direct-Zol™ RNA Miniprep kit (R2051) following the manufacturer’s protocol. Total RNA was reversely transcribed using a miScript II RT Kit (218161 Qiagen, Hilden, Germany), while mRNA templates were reversely transcribed using the Moloney Murine Leukemia Virus (M-MLV RT) reverse transcriptase by Promega (9PIM170; Promega, Madison, WI, USA). RT-PCR was performed using SYBR green, carried out on a Bio-Rad CFX96 Real-Time System. For validation of transfection efficiency in cell culture experiments, miR-337-3p pre-designed miRCURY LNA Uni RT primer mix (339306, Qiagen) was used. Consequently, total RNA was reversely transcribed using its compatible miRCURY LNA™ Universal cDNA synthesis kit II (203301, Qiagen) followed by real-time PCR with the LNA enhanced primers using ExiLENT SYBR green (203401, Thermofisher, Waltham, MA, USA). Quantification of transcript level by RT-PCR was done by using the relative Ct (ΔΔCt) method. mRNA transcripts were normalized to L7, while miR transcripts were normalized to U6 or 5s (pre-designed miRCURY LNA Uni RT primer mix, Qiagen). The choice of reference genes was based on their stability in the different samples analyzed. The sequence of the primers used in this study can be found in Table 1.

Total RNA was extracted from 50 mg of each tissue sample (liver) using the commercial kit E.Z.N.A ^® Total RNA Kit I (Omega) and Rnase-free Dnase I, following the manufacturer’s guidelines. Then the total RNA pellet was dissolved in diethylpyrocarbonate water, quantified by spectrophotometry (NanoDrop Technologies), and its quality was evaluated using 1% agarose gel. Subsequently, 1 μg of total RNA was used as a template for cDNA synthesis, using MMLV-RT reverse transcriptase (Promega) and oligo-dT primer (Invitrogen), according to the standard procedure (10 (link)).

Liver and brain tissues from E. maclovinus at different experimental conditions were aseptically extracted and used for total RNA extraction. RNA was extracted from each tissue (50 mg) by homogenization in TRIzol (Ambion) following the manufacturer’s instructions. The RNA pellets were dissolved in diethyl pyrocarbonate water and stored at −80°C. Subsequently, the RNA was quantified at 260 nm on a NanoDrop spectrophotometer (NanoDrop Technologies^®), and their quality determined by electrophoresis on 1% agarose gel. Total RNA (2 µg) was used as a reverse transcription template to synthesize cDNA, applying MMLV-RT reverse transcriptase (Promega) and the oligo-dT primer (Invitrogen) according to standard procedures.

In order to perform a technical validation of DEG analysis, we designed primers using the software Primer 3 Plus (http://www.bioin formatics.nl/cgi-bin/primer3plus/primer3plus.cgi) based on the sequences generated by the de novo assembly. First, from the same input used to construct the libraries, total RNA was treated with DNase I (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA) according to the standard protocol. The first strand cDNA was obtained by reverse transcription using the system MMLV-RT reverse transcriptase (Promega, Madison, WI, USA) and oligo dT primers. The cDNA concentration was obtained by measuring absorbance at 260 nm. Each cDNA sample was diluted to 50 ng*μL^− 1 before being used in qRT-PCR assays. The qRT-PCR assay was performed using a LightCycler® 96 Real-Time PCR kit (Roche Diagnostics, Mannheim, Germany) with LC-FastStart DNA Master SYBR Green I to measure the DNA product derived from RNA, as previously described by García-Rojas and colleages [80 (link)]. Additionally, to validate the gene expression profiles in the selected subclusters, qRT-PCR of one gene belonging to each subcluster (4, 7 and 11) was performed using total RNA from five independent fruits at 150, 240, 300 and 390 DAFS. All qRT-PCR were performed using five biological replicates and the gene expression values were normalized relative to the PamActin gene (GenBank JN786942).

In the case of qPCR assays which required the synthesis of cDNA, the reverse transcription was accomplished in 2 μg of total RNA as template using a MMLV-RT reverse transcriptase with previously primed oligo dT (Promega, Madison, WI), according to the manufacturer instructions. The obtained cDNA was then stored at − 20 °C until further qPCR assays.