iPSNs were harvested in 1 × DPBS with calcium and magnesium and pelleted using a microcentrifuge. 350 μL RLT Buffer was added to iPSN pellets and RNA was isolated using the RNeasy kit (QIAGEN). RNA concentrations were determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). For detection of G4C2 and G2C4 repeat containing transcripts, 1 μg RNA was used for cDNA synthesis using gene specific primers and the Superscript IV First-Strand cDNA Synthesis System (Thermo Fisher Scientific). For detection of TDP-43 mRNA targets, 1 μg RNA was used for cDNA synthesis using random hexamers and the Superscript IV First-Strand cDNA Synthesis System (Thermo Fisher Scientific). All qRT-qPCR reactions were carried out using SYBR Green Master Mix or TaqMan Gene Expression Master Mix (Thermo Fisher) and an Applied Biosystems QuantStudio 3 (Applied Biosystems). Previously described primer/probe sets (see Supplemental Table 2 for sequences) [24 (link)] were used to detect G4C2 and G2C4 repeat containing transcripts. Previously described primer sets (see Supplemental Table 2 for sequences) [30 (link), 32 (link), 40 (link), 43 (link)] were used to detect truncated STMN2 and cryptic exon containing mRNA transcripts. TaqMan Gene Expression Assays (see Supplemental Table 2 for probe information) were used to detect mRNA targets. GAPDH was used for normalization of gene expression.
Superscript 4 first strand cdna synthesis system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Superscript IV First-Strand cDNA Synthesis System is a reagent kit for the reverse transcription of RNA into complementary DNA (cDNA). The system includes the SuperScript IV Reverse Transcriptase enzyme and other necessary components for the cDNA synthesis reaction.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Allprep dna rna protein mini kit, by Qiagen (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Qiaamp viral rna extraction protocol, by Qiagen (1 mentions)
Klenow fragment, by New England Biolabs (1 mentions)
Ampure xp bead purification, by Beckman Coulter (1 mentions)
Qx200 ddpcr system, by Bio-Rad (1 mentions)
Rq1 rnase free dnase 1, by Promega (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Cfx manager software, by Bio-Rad (1 mentions)
9 protocols using superscript 4 first strand cdna synthesis system
1
RNA Isolation and qRT-PCR Analysis of Neuronal Transcripts
2
Quantitative Analysis of C9orf72 and Nup Transcripts
3
Real-Time PCR Gene Expression Analysis
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Total RNA was extracted from samples using the AllPrep DNA/RNA/Protein Mini Kit (#80004, Qiagen Sciences, Inc., Germantown, MD, United States) according to the manufacturer’s protocol. RNA was eluted with RNase-free water. Total RNA was quantified using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). For reverse transcription, 0.25 μg of total RNA was used in the SuperScriptTM IV First-Strand cDNA Synthesis System (#18091050, Thermo Fisher Scientific, Waltham, MA, United States) with a mixture of 50 μM oligo-dT and 50 ng/μl random hexamers as primers. Real-time primers were designed with Primer3web, version 4.4.01 and validated using melt curve analysis and agarose gel electrophoresis. PCR primers used in this study are shown in Supplementary Table 1 . Significant gene expression was evaluated using the FastStart Universal SYBR Green Master (ROX; #4913850001, Roche Molecular Systems, Inc., Branchburg, NJ, United States) in triplicate in QuantStudioTM 3 Real-Time PCR Systems (Thermo Fisher Scientific, Waltham, MA, United States). The relative quantity of mRNA was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the comparative 2–ΔΔCt method.
4
Real-Time Quantitative PCR Analysis
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Real-time (RT)-PCR was performed as described previously (Zhou et al., 2020b) . In brief, total RNA was extracted from samples using the AllPrep DNA/RNA/Protein Mini Kit (80004, Qiagen Sciences, Inc.) and 0.25 µg of total RNA was reverse transcribed by SuperScript TM IV First-Strand cDNA Synthesis System (18091050, Thermo Fisher Scientific) with a mixture of oligo dT and random hexmer. Amplification of cDNA was performed with optimized PCR primers designed by Primer3 software (https://bioinfo.ut.ee/primer3/) and synthesized by Integrated DNA Technologies (Coralville, Iowa) in SYBR Green master (Rox) system (04913850001, Roche, Basel, Switzerland) on QuantStudio TM 3 Real-Time PCR Systems (ThermoFisher). Detailed information on PCR primers used in this study are provided in Supplemental Table 1. The relative quantity of mRNA was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the comparative 2 -∆∆Ct method.
5
Hamster Swab and Tissue cDNA Synthesis
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Complementary DNAs (cDNAs) were prepared according to Briese et al. (48 (link)). Briefly, RNA was extracted from hamster swabs and tissues following the QiaAmp Viral RNA extraction protocol (Qiagen), and 11 μl was taken into the SuperScript IV First-Strand cDNA synthesis system (Thermo Fisher Scientific) following the manufacturer’s instructions. After ribonuclease H treatment, second-strand synthesis was performed using Klenow fragment (New England Biolabs) following the manufacturer’s instructions. The resulting double-stranded cDNAs (ds-cDNA) were then purified using Ampure XP bead purification (Beckman Coulter) and eluted into 30 μl of water.
6
Isolation and Analysis of Neuronal RNA
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DRG neurons were grown in tripartite microfluidic chambers with two microgroove barriers (200 and 500 μm, respectively; illustrated in Supplementary Figure S3A ). The design of the tripartite chambers makes sure that no neuronal soma will be present in the distal axon compartment. TRIzol reagent (Thermo Fisher Scientific) was applied to either soma or distal axon compartment (50 μl per compartment). Lysates from 10 chambers were pooled together and total RNA was extracted following the manufacturer’s protocol. After first-strand cDNA was synthesized using SuperScript IV First-Strand cDNA Synthesis System (Thermo Fisher Scientific), polymerase chain reaction (PCR) was performed with specific primers for mouse clones as following:
Fto, 5′-GAAGTGGTGAGGATCCAAGG-3′ and 5′-CTGCCTTCGAAGCTGGACTC-3′;
β-actin, 5′- AGGGAAATCGTGCGTGACAT-3′ and 5′-ACGCAGCTCAGTAACAGTCC-3′; Nrxn2, 5′- CAGACCTCATCGCTGACGC-3′ and 5′- AGGCGGTCCATCCGTGTAC-3′; H1f0, 5′-AGTATATCAAGAGCCACTACAAGG-3′ and 5′-AATGTATTTACAGAAAACAGGAGG-3′.
Fto, 5′-GAAGTGGTGAGGATCCAAGG-3′ and 5′-CTGCCTTCGAAGCTGGACTC-3′;
β-actin, 5′- AGGGAAATCGTGCGTGACAT-3′ and 5′-ACGCAGCTCAGTAACAGTCC-3′; Nrxn2, 5′- CAGACCTCATCGCTGACGC-3′ and 5′- AGGCGGTCCATCCGTGTAC-3′; H1f0, 5′-AGTATATCAAGAGCCACTACAAGG-3′ and 5′-AATGTATTTACAGAAAACAGGAGG-3′.
7
Quantification of Gene Expression by ddPCR
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Isolated total RNA from stretched and non-stretched human SC cells was reverse transcribed to cDNA using the SuperScript IV First-Strand cDNA Synthesis System (Thermo Fisher Scientific) according to the manufacturer's recommended protocols. Droplet digital polymerase chain reaction (ddPCR) EvaGreen-based expression assays for specific human genes were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA) (Table 2 ). The Bio-Rad QX200 ddPCR system was used to generate and measure the partitioned droplets for absolute quantification (copies/20 µl) of target genes as previously described.30 (link)
GAPDH and HPRT1 were used as the internal reference genes to normalize the gene expression across different samples.
GAPDH and HPRT1 were used as the internal reference genes to normalize the gene expression across different samples.
8
Transcriptome Analysis of Drosophila Larvae
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Groups of 1–3 h L3 larvae were homogenized in 500 μL TRIzol (Invitrogen #15596018) and RNA was extracted according to manufacturer’s protocol. RNA extracts were treated with RQ1 RNase-free DNAse I (Promega #M6101) according to manufacturer’s protocol to remove any genomic DNA contaminants. cDNA was prepared using the SuperScript IV First Strand cDNA Synthesis system (Thermo Fisher Scientific #18091200) with 1 μg total RNA and using oligo(dT)20 primers, following the kit protocol with an annealing temperature of 52.5°C. qPCR was performed on a Roche LightCycler 480 II using the LightCycler 480 SYBR Green I Master mix (Roche #18887320), 500 nM final primer concentration, and 1:25 final dilution of cDNA. The following qPCR primers were used:
For dmyc primers, qPCR reactions had an annealing temperature of 65°C; for other primers, the annealing temperature was 60°C.
For dmyc primers, qPCR reactions had an annealing temperature of 65°C; for other primers, the annealing temperature was 60°C.
9
RNA Isolation and cDNA Synthesis for Kidney and Embryo
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Freshly dissected adult kidneys and pools of 20 embryos/larvae (from 6 hpf to 5 dpf, raised in E3 medium) were sonicated in TRIzol for 10 s at 30 W (Bioruptor, Diagenode) for homogenisation. Isolation of total RNA was performed using TRIzol (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized using SuperScript IV First-Strand cDNA Synthesis System (ThermoFisher Scientific) according to manufacturer’s instructions. Endpoint RT-PCR was performed according to the manufacturer’s protocol using My-Budget PCR master mix (Bio-Budget), for primers used see Table S1 .
RT-qPCR was performed according to the manufacturer’s protocol using primaQUANT qPCR-SYBR-Green master mix (Steinbrenner-Laborsysteme). No template control (NTC) and no reverse trancriptase (NRT) samples were included as negative controls. At least three biological replicates and two technical replicates of qPCR reactions were performed. qPCR data were obtained using the Bio-Rad CFX Manager software (Bio-Rad). For primers used see TableS1 .
RT-qPCR was performed according to the manufacturer’s protocol using primaQUANT qPCR-SYBR-Green master mix (Steinbrenner-Laborsysteme). No template control (NTC) and no reverse trancriptase (NRT) samples were included as negative controls. At least three biological replicates and two technical replicates of qPCR reactions were performed. qPCR data were obtained using the Bio-Rad CFX Manager software (Bio-Rad). For primers used see Table
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