Quantitative real-time PCR Total RNA was prepared using PeqGOLD RNApure (Peqlab) according to the manufacturer’s protocol. 500 ng RNA was reverse transcribed using the Protoscript II cDNA Synthesis Kit (NEB, procedure according to manufacturer’s protocol). Quantitative PCR analysis was conducted using the Fast Start SybrGreen MasterMix according to the standard protocol (Roche). Please find Primer sequences for real-time PCR (gene symbols and species followed by sequences of forward and reverse primers) in Supplementary Table S3 .
Protoscript 2 cdna synthesis kit
Manufactured by New England Biolabs
Sourced in United States
The ProtoScript II cDNA Synthesis Kit is a laboratory product designed for the synthesis of complementary DNA (cDNA) from RNA templates. The kit includes the necessary reagents and enzymes to perform this reaction.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy plus universal kit, by Qiagen (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Peqgold rnapure, by Avantor (1 mentions)
Faststart sybr green master mix, by Roche (1 mentions)
Rneasy mini kit, by New England Biolabs (1 mentions)
Onetaq 2x master mix, by New England Biolabs (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Nebnext ultra 2 directional rna library prep kit, by Illumina (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green supermix, by Bio-Rad (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Single cell rna purification kit, by Norgen Biotek (1 mentions)
Onetaq dna polymerase, by New England Biolabs (1 mentions)
12 protocols using protoscript 2 cdna synthesis kit
1
Quantitative Real-Time PCR Assay
2
Quantitative Analysis of CAPN3 and PGC1α Expression
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QIAzol lysis reagent was used to isolate RNA from select tissues. Total RNA was DNase treated and purified using the RNeasy Plus Universal Kit (QIAGEN). First-strand DNA was synthesized with the ProtoScript II cDNA Synthesis Kit (New England Biolabs). qRT-PCR for CAPN3 and PGC1α was performed using the primer sets listed in Table S15 . The 2–ΔΔCt method was used for calculating relative expression. Expression data were normalized to mouse GAPDH mRNA levels and are shown as means of relative expression values obtained from duplicate samples and normalized to WT control levels (set at 1) in conjunction with the standard error of the mean and are presented in a graph format.
3
Voriconazole Induction of Cyp51 Genes
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A modification of a previously-described protocol for voriconazole treatment was used to generate mycelia for qRT-PCR analysis.23 (link) Briefly, 3 × 107 conidia of the wild type strain and 6 × 107 conidia of the ΔramA strain were inoculated into GMM+YE broth and incubated at 37°C. After 12 hr incubation, a concentration of voriconazole solubilized in DMSO equal to 50% of the MIC for each strain as determined by E-test assay (Af293 = 1 μg/ml; ΔramA = 3 μg/ml) was added to the treatment cultures. An equal volume of DMSO vehicle was added to control cultures of each strain. Incubation was continued at 37°C for 6 hr. Extraction of total RNA and synthesis of cDNA was performed using the Qiagen RNEasy Mini Kit and New England Biolabs ProtoScript II cDNA Synthesis Kit, respectively.
Biological triplicates of each treatment condition were analyzed by quantitative real-time PCR (qRT-PCR) in technical triplicate using A. fumigatus tubA (tubulin) as the endogenous standard. Target amplification primers for cyp51A (P12/ P13) and cyp51B (P14/P15) were as described previously.39 (link) The qRT-PCR analyses were conducted on an Applied Biosystems StepOne Real-Time PCR system, and data were analyzed by relative quantitation to wild type DMSO transcription levels using the ΔΔCT method (StepOne Software, v2.2.2).
Biological triplicates of each treatment condition were analyzed by quantitative real-time PCR (qRT-PCR) in technical triplicate using A. fumigatus tubA (tubulin) as the endogenous standard. Target amplification primers for cyp51A (P12/ P13) and cyp51B (P14/P15) were as described previously.39 (link) The qRT-PCR analyses were conducted on an Applied Biosystems StepOne Real-Time PCR system, and data were analyzed by relative quantitation to wild type DMSO transcription levels using the ΔΔCT method (StepOne Software, v2.2.2).
4
RT-PCR and RT-qPCR Protocols for Gene Expression Analysis
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RT was performed on DNase I treated RNA using the Protoscript II cDNA synthesis kit (NEB) with random primers. Semi-quantitative RT-PCR was carried out using OneTaq 2X mastermix (NEB) according to the manufacturer's instructions. RT-PCR products were resolved in 2% agarose TAE gels stained with ethidium bromide. RT-qPCR was carried out using iTaq Universal SYBR Green Supermix on the CFX96 qPCR platform. Primers used in RT-PCR and RT-qPCR are listed in Supplementary Table S1 .
5
Quantitative Expression Analysis of APOE in C. elegans
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Worms were washed from near-starved plates (3 plates for WT and integrated, 9 plates for extrachromosomal) and pelleted. Each pellet was flash frozen in liquid nitrogen, thawed on ice, and vortexed 6 times. The pellet was further disrupted by needle and syringe in RNA extraction buffer. RNA was isolated (Zymo Quick-RNA Miniprep; QIAGEN RNeasy Mini Kit), and 1ug was reverse transcribed into cDNA with the Protoscript II cDNA synthesis kit (NEB). qPCR was performed using SYBR Green (FisherSci) on a Roche 480 Lightcycler (Roche) using primers: APOE forward 5′-CCTGGACGAGGTGAAGGAGCA-3′, reverse 5′-CTCGAACCAGCTCTTGAGGC-3′ and tba-1 forward 5′-ATCTCTGCTGACAAGGCTTAC-3′, reverse 5′-GTACAAGAGGCAAACAGCCAT-3′. Ct values were exported and analyzed using the ΔCt method. No values were detected for no template and no RT controls. Data represents the mean of two biological replicates with three technical replicates each. The housekeeping gene tba-1 was used as a control, and relative expression was normalized to this reference gene.
6
In vitro Translation Sample Processing
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For in vitro translation, post-translation samples (20 μL) were treated with DNase I (0.1 U/μL) in DNase reaction buffer (10 mM Tris-HCl pH 7.6, 2.5 mM MgCl2, 0.5 mM CaCl2) at 37°C for 30 minutes. The reaction was quenched by 1 μL of 0.5 M EDTA, followed by heat inactivation at 75°C for 10 minutes. The DNA-free samples were diluted 10-fold, and 2 μL of each diluted sample was used to synthesize cDNA by ProtoScript II cDNA Synthesis Kit (NEB). The reaction was incubated at 25°C for 5 minutes, followed by 42°C for 1 hour and 80°C for 5 minutes. The cDNA was diluted 100-fold before qPCR measurement. The qPCR sample (20 μL) contained 1 μL 100-fold diluted cDNA, 10 μL SYBR Green Supermix (NEB), and 250 nM primers (see Supplementary Table 2). The data points of cycles were plotted in Fig. 6B .
7
Transcriptomic Profiling of mESCs
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RNA was extracted from mESC and EBs using an RNEasy Kit (QIAGEN) according to the manufacturer’s recommendations and filtered pipette tips. After extraction samples were quantified with Qubit (Thermo Scientific). RT was performed using the ProtoScriptII cDNA Synthesis Kit (NEB) to give cDNA. For qPCR, cDNA from each sample was ran in triplicate with primers designed to amplify 180–220 bp spanning exons of each target gene of interest on a StepOne™ Real-Time PCR System (Applied Biosystems), and GAPDH was ran in triplicate for each sample to normalize expression. For mRNA sequencing of wild-type and Cbx2 KO (with OHT for two days) mESCs, 1.0 μg of total RNA per sample was used to generate sequencing libraries with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®. following manufacturer’s instructions. The library was then quantified by using Qubit dsDNA HS Assay kit and examined using a TapeStation High Sensitivity DNA assay kit. The prepared mRNA library was then sequenced on Nextseq 500 to generate ~ 20 million PE75 reads.
8
In Vitro Translation mRNA Analysis
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For in vitro translation, post-translation samples (20 µL) were treated with DNase I (0.1 U/µL) in DNase reaction buffer (10 mM Tris-HCl pH 7.6, 2.5 mM MgCl2, 0.5 mM CaCl2) at 37 °C for 30 min. The reaction was quenched by 1 μL of 0.5 M EDTA, followed by heat inactivation at 75 °C for 10 min. The DNA-free samples were diluted tenfold, and 2 μL of each diluted sample was used to synthesize cDNA by ProtoScript II cDNA Synthesis Kit (NEB). The reaction was incubated at 25 °C for 5 min, followed by 42 °C for 1 h and 80 °C for 5 min. The cDNA was diluted 100-fold before qPCR measurement. The qPCR sample (20 µL) contained 1 μL 100-fold diluted cDNA, 10 μL SYBR Green Supermix (BioRad), and 250 nM primers (see Supplementary Table 2 ).
9
Relative Gene Expression Analysis via RT-qPCR
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RT-qPCR was used to determine the relative gene expression of E-cadherin, Bcl-2, YAP, Ki-67 and Cyclin-D1 in Y-27632- and TTNPB-treated cells. First, total RNA was extracted using the Single Cell RNA Purification Kit (Norgen Biotek Corp.) according to the manufacturer’s instructions. RNA concentrations were then determined by spectrophotometer. The RNA was transcribed into cDNA using the protoscript II c-DNA synthesis kit (New England Biolabs). cDNA generation consisted of the following steps: 65 °C for 5 min, 42 °C for 60 min, and 80 °C for 5 min. Finally, fast SYBR™ Green Master Mix (Thermo Fisher Scientific) was used for the RT-qPCR gene expression analysis (performed on the Applied Biosystems™ StepOnePlus™ Real-Time PCR System). The mRNA expression levels were all quantified relative to the housekeeping gene, GAPDH.
10
Quantitative Real-Time PCR Analysis of Mitochondrial Genes
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Total RNA was isolated from muscles tissues using the QIAzol lysis reagent, treated with DNase and purified using RNeasy Plus Universal Kit (Qiagen). ProtoScript II cDNA Synthesis Kit (New England Biolabs) was used for first-strand DNA synthesis. Primer sets for PGC-1α, Cox1, Cox3 and Atp5d were obtained from previous publications33–35 (link) (details in the Supplementary data ).
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