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Goscript reverse transcriptase system

Manufactured by Promega
Sourced in United States

The GoScript Reverse Transcriptase System is a laboratory tool designed for the synthesis of complementary DNA (cDNA) from RNA templates. It provides a reliable and efficient means of converting RNA into cDNA for downstream applications such as gene expression analysis, PCR, and sequencing.

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65 protocols using goscript reverse transcriptase system

1

Quantifying mRNA Levels with CGG Repeats

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For assessment of the level of mRNAs containing CGG repeats, transfection of COS7 with 100×CGG construct and ASOs and total mRNA isolation was performed as described in “miRNA level quantification”. Next, cDNA was synthesized with the use of GoScriptTM Reverse Transcriptase System (Promega) according to the manufacturer’s instructions with the exception of the first-strand synthesis reaction temperature which was 37 °C. We used an anchored oligo(dT) primer containing oligo dT tract which allowed for exclusive reverse transcription of polyA+ RNAs. qPCR was performed with the use of iTaq™ Universal SYBR® Green Supermix (Bio-Rad) according to manufacturer’s instructions and analyzed with the use of QuantStudio 7 Flex Real-Time PCR System machine (Thermo Fisher Scientific). To ensure amplification of 100×CGG construct mRNA but not DNA following primers were used: forward primer complementary to the 3′ part of GFP sequence and universal reverse (UR) primer. Primers for amplification of endogenous mRNA of FMR1 and NUB1 were set in 3′UTR of transcripts. Ct values were normalized against GAPDH and amplified with gene-specific forward primer set in 3′ part of mRNA and UR primer. All primers are listed in Supplementary Table S2. Fold differences in expression level were calculated according to the 2−ΔΔCt method.
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2

SAM68-dependent alternative splicing analysis of CGG expansions

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For SAM68-dependent alternative splicing analysis upon CGGexp expression, we used COS7 cells, 24-well plate, 300 ng of either 20×GG or 60×CGG or 100×CGG construct, 100 ng of SMN2 minigene and 100 ng of Atp2a1 minigene per well and 11-nucleotide-long ASOs at 100 or 200 nM concentration. After 48 h, cells were harvested using TRI Reagent (Merck), and the total RNA was isolated according to the manufacturer’s instructions. cDNA was synthesized with the use of GoScriptTM Reverse Transcriptase System (Promega) and random primers (Promega) according to the manufacturer’s instructions. PCR was performed with GoTaq DNA Polymerase and primers listed in Supplementary Table S2. Sequences of SMN2_F and SMN2_R primers were obtained from13 (link). PCR products were separated in 1–2% agarose gel with ethidium bromide. Images were captured with the use of G:Box EF2 (Syngene). Signals of PCR products were assessed with the use of GeneTools (Syngene). Percent of alternative exon inclusion (PSI, the percent of alternative exon spliced in) was calculated based on signals of two bands according to the following formula (isoform with included exon*100)/(isoforms with included exon + excluded exon).
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3

Quantitative Real-Time PCR Protocol

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Total RNA was extracted from the cells by using Direct-zol RNA Miniprep Plus Kit (Zymo Research, USA). Total RNA and purity of RNA were quantified and determined using NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA). Desired amount of total RNA was converted into first-strand cDNA GoScriptTM Reverse Transcriptase System (Promega) according to manufacturer’s protocol. cDNA was amplified on a thermal cycler (BioRad Laboratories, USA) with annealing step at 25 °C for 5 min, extending step at 42 °C for up to 1 h and the reaction was stopped at by inactivating the reverse transcriptase at 70 °C for 15 min. Synthesized cDNA was mixed to the specific forward and reverse primers (Additional file 1: Table S1) and Luna Universal qPCR Master Mix (NEB, USA). Real-time polymerase chain reaction (RT-PCR) was initiated using Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Massachusetts, USA). The amplification program was 95 °C for 5 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 10 s. Reactions were run in duplicate in at least three independent experiments. The results were normalized to the expression of GAPDH. Gene expression was analyzed using 2−ΔCT and 2−ΔΔCT methods [19 (link)].
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4

Mosquito Dissection, RNA Extraction, and cDNA Synthesis

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Dissections, RNA extraction, and cDNA synthesis were performed as described in Calkins et al. [11 (link)]. In brief, mosquitoes were anesthetized on ice before being dissected in mosquito Ringer solution (consisting of 150 mM NaCl, 3.4 mM KCl, 1.7 mM CaCl2, 1.8 mM NaHCO3, 1 mM MgCl2, 5 mM Glucose, 25 mM HEPES; pH 7.1). Tissues were isolated, transferred to 1.5-ml micro-centrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA), and preserved in TRIzol® reagent at −80 °C until utilized in RNA isolation. Total RNA was isolated using the method of Chomczynski and Sacchi [17 (link)] and quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). cDNA libraries were synthesized using 4 µg of total RNA and the GoScriptTM Reverse Transcriptase system with random primers (Promega, Madison, WI, USA), following manufacturer’s protocols. cDNA libraries were stored at −20 °C until needed for qPCR.
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5

TRPV4 Channel Expression Analysis

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Extracted RNA was reverse-transcribed into cDNA using the Goscript
TM Reverse Transcriptase System (Promega, Madison, USA) according to the manufacturer’s instructions. Semi-quantitative PCR was performed using a PCR kit (Tiangen, Beijing, China) on a thermo-cycler (Bio-Rad, Hercules, USA). PCR reactions were performed in a total volume of 25 μL containing 1 μL cDNA. Primers of the TRPV4 channel were designed for semi-quantitative PCR. All specific primers for the TRPV4 channel were designed based on Genbank mouse sequences. Specific primer sequences are as follows: TRPV4 channel, sense: 5′-TACGACCTGCTGCTTCTCAA-3′, antisense: 5′-TCCTCATCTGTCACCTCACG-3′; and GAPDH, sense: 5′-GGTGAAGGTCGGTGTGAACG-3′, antisense: 5′-CTCGCTCCTGGAAGATGGTG-3′. The cDNA product was PCR amplified using specific primers for the TRPV4 channel. The PCR annealing temperature for each primer pair was optimized using a Master Cycler Gradient Thermocycler (Eppendorf, Hamburg, Germany). PCR reaction consisted of 30 s denaturation at 94°C, 30 s for annealing at 58–64°C, 30 s for elongation at 72°C, and 30 amplification cycles. PCR products (15 μL) were separated by electrophoresis on a 1% agarose gel in Tris-borate/EDTA buffer. The quantification of semi-quantitative PCR was performed by using the optical density (OD) method.
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6

Quantifying uhrf1 Expression in Wildtype and hmx1 Knockout Zebrafish

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For the analysis of uhrf1 expression, wildtype and hmx1 knockout embryos at 24 hpf and 5 dpf were euthanized. For whole body transcript analysis at 24 hpf and 5 dpf, 60 embryos obtained from different breeding were pooled together. For eye transcript analysis, both eyes were isolated from 20 embryos at 5 dpf and pooled together. All experiments were repeated three times.
First-strand cDNA synthesis was performed using the AffinityScript Multiple Temperature Reverse Transcriptase kit (Agilent, Basel, Switzerland) according the manufacturer’s protocol. cDNA was generated (GoScript Reverse Transcriptase System; Promega) and Real time PCR (FastStart SYBR Green Master Roche) was performed using uhrf1-F 5’-TCCAGGAGTCCAAGAGAGGAA-3’ and uhrf1-R 5’-TCTGCTGAACACAGTTCGGG-3’; dnmt1-F 5’-TTACTTTGGGCAAGAGGAGAGC-3’, dnmt1-R 5’-GACACCACACCGTTGTCTCT-3’; hmx1-F 5’-CGAAACCTCCAGGAG TCCAAG-3’, hmx1-R 5’-CGGGTCTTTTTCTTTCGGGC-3’.
Gene expression change was determined using the 2–ΔΔCt method; relative values were normalized with β-actin.
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7

Quantitative Real-Time PCR Verification

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Microarray data were verified by quantitative real time PCR. Briefly, First-strand cDNA was synthesised using random hexamer primers and the GoScript Reverse Transcriptase System (Promega), and qPCR was performed using the SYBR green PCR protocol (Applied Biosystems) on the Chromo4 real-time PCR system (Bio-Rad). Primer sequences are presented in S5 Table. Each experiment included ‘no template’ controls, was run in duplicate and had an 18S RNA control. Each independent experiment was repeated three times, and the results were analysed by independent-samples t-test.
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8

Quantitative Gene Expression Analysis

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RNA for gene expression analysis from EndoC-βH1 was extracted using TRIzol reagent (Invitrogen) and synthesized into complementary DNA using the GoScript Reverse Transcriptase System (Promega). RNA for gene expression analysis from primary human pseudoislets was extracted using the PicoPure RNA isolation kit (Life Technologies), and cDNA was synthesized using the Maxima first strand cDNA synthesis kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Quantitative qPCR (qPCR) was performed using TaqMan real-time PCR assays on a 7900HT Fast Real-Time PCR System (all Applied Biosystems, Supplementary Table 4). Ct-values were analyzed using the ΔΔCt method, and target genes were normalized to the combined average of the housekeeping PPIA, GAPDH and TBP. CALCOCO2 expression in EndoC-βH1 and primary islets was extracted from previously published and analyzed RNA-seq data71 (link),72 (link).
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9

RNA Extraction and qPCR Analysis Protocol

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Cells were harvested, and total RNA was isolated using the PureLink™ RNA Mini Kit (Thermo Fisher Scientific, Cat. No. 12183020) as per the manufacturer’s instructions. First-strand cDNA synthesis of 250–500 ng total RNA was done using GoScript Reverse Transcriptase System (Promega, Catalog No. A5004). Relative mRNA expression of target genes was checked using SYBR® Green Real-Time qPCR Master Mix (Thermo Fisher Scientific, Cat. No. 4309155) in QuantStudio™ 7 Flex Real-Time PCR System, 96-well block (Applied Biosystems™, Cat. No. 4485701). The primer sequences used are listed in Table 1.
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10

Total RNA Extraction and qRT-PCR Analysis

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Total RNA was extracted using a ReliaPrep™ RNA Cell Miniprep System (Promega) and quantified by a Nanodrop 2000 (Thermo, Wilmington, DE, USA). For RT-PCR, 0.5 μg of total RNA from each sample was used for cDNA synthesis using the GoScript™ Reverse Transcriptase system (Promega). Real-time PCR was performed on a real-time PCR system (7500 Fast Machine, Applied Biosystems, Thermo) using GoTaq® qPCR Master Mix (Promega). The comparative Ct (∆∆Ct) method was used to calculate the gene expression levels after normalization, according to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The data show the relative values to the mRNA levels when cultured on control tissue culture plastic. The primers and their sequences are shown in Table 1.
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