Luciferase mrna

RNA was isolated from frozen testes from 8, 80-d-old LCARKO and 8 control mice, or 6 Adipo-ARKO or 6 control mice, using the RNeasy Mini extraction kit with RNase-free DNase on the column digestion kit (QIAGEN) according to the manufacturer’s instructions. For quantitative RT-PCR, 5 ng Luciferase mRNA (Promega Corporation, Madison, WI, USA) was added to each testis sample before RNA extraction as an external standard (41 (link)). RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Random hexamer-primed cDNA was prepared using the SuperScript VILO cDNA Synthesis Kit (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. Quantitative PCR was performed on 80-d-old LCARKO and control testes for the genes listed in Table 2, using an ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and the Roche Universal Probe library (Welwyn, United Kingdom), as described previously (30 (link)). The expression of each gene was related to an external positive control luciferase, as published previously (42 (link)), and all genes were expressed per testis.

Luciferase mRNA (Promega) was used as the exogenous internal reference (ref mRNA) to estimate the percentage of RNA loss during extraction. After the centrifugation of the lysed cells, 1 μL of a range of 1 × 10¹¹ to 1 × 10⁷ref mRNA transcripts/μL were added to the aqueous phase. After RNA extraction, the amount of ref mRNA recovered from samples was measured by qPCR and the standard curve generated from serial dilutions of ref mRNA. The recovery fraction was calculated by dividing the determined levels by the initial number of ref mRNA transcripts added to the samples.

Total RNA was isolated from the groups of oocytes with the RNeasy Micro Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. Five picograms of luciferase mRNA (Promega, The Netherlands) were added to each group prior to RNA isolation to account for RNA loss during the isolation process. During the procedure, RNA was treated with DNase I to exclude any potential genomic DNA contamination. Isolated RNA was eluted in 12 μL RNase-free water and immediately used for reverse transcription polymerase chain reaction (RT-PCR). Reverse transcription was performed in a final volume of 20 μL, consisting of 75 mM KCl, 50 mM Tris- HCl (pH 8.3), 5 mM DTT, 3 mM MgCl₂, 1 mM dNTPs, 2.5 μM random hexamer primers, 20 U RNase OUT and 100 U SuperScript III RT (all purchased at Invitrogen Corporation, Carlsbad, CA, USA). The reaction tubes were incubated at 25 °C for 10 min, at 42 °C for 1 h and at 70 °C for 15 min to inactivate the reaction. One tube without RNA and one with RNA, but without reverse transcriptase, were analyzed as negative controls. To quantify the mRNA recovery rate, 5 pg of luciferase mRNA (not subjected to RNA isolation) were subjected to cDNA synthesis as well.

Testis RNA was extracted using Trizol (Life Technologies, Paisley, UK) and levels of specific mRNA species were measured by real-time PCR. Total RNA was reverse transcribed using random hexamers and Moloney murine leukaemia virus reverse transcriptase (Superscript III, Fisher Scientific UK Ltd., Loughborough, UK) as described previously [25 (link)]. To normalise the data, external standard (luciferase mRNA: Promega UK, Southampton, UK) was added to each testis at the start of the RNA extraction [26 (link)]. For real-time PCR the SYBR green method was used with SYBR mastermix (Agilent Technologies, Wokingham, UK) [27 (link)]. All primers were designed by Primer Express 2.0 (Applied Biosystems, Warrington, UK) or Primer-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using parameters previously described [28 (link)] and the primers used are described in Additional file 2. Transcript levels were normalised to the luciferase external standard, which generates a relative value of transcript expression per testis [29 (link)].

In vitro translation rates represent the increase in luminescence as a function of time using a standard rabbit reticulocyte lysate system (Promega) with luciferase mRNA (Promega). Manufacturer’s instructions were followed with a few modifications. Briefly, each reaction (30 μl) contains 12.6 μl of rabbit reticulocyte lysate, 0.5 μl of luciferase mRNA (1 mg/ml), 0.3 μl of amino acid mixture minus leucine (1 mM), 0.3 μl of amino acid mixture minus methionine (1 mM), 2 μl of TEV protease (15 μM), and 14.3 μl of 5 μM protein (MBP-FUS/FMRP) or buffer [25 mM sodium phosphate (pH 7.4), 50 mM KCl, and 2 mM DTT). First, the reaction was incubated for 10 min, and then, end-point luminescence measurements were carried out in intervals of 10 min up to 50 min. Each end-point luminescence measurement contained 75 μl of luciferase substrate mixed with 2.5 μl of unpurified translation mixture measured in a white opaque 96-well plate (Corning 3990). A SpectraMax i3× multimode plate reader (Molecular Devices) at 25°C was used to detect the luminescence. The translation rates represent the line of best fit from the end-point luminescence readings as a function of time.