Isolation of total RNA from the gastrocnemius (GA) was performed using an RNeasy Fibrous Tissue Mini Kit (Qiagen Catalog # 74704) according to the manufacturer’s instructions. Skeletal muscle RNA was treated with an RNase-free DNase kit (RNase-Free DNase Set, Qiagen Catalog # 79254) in column according to the manufacturer’s instructions. First strand cDNA was synthesized using 1 μg of purified RNA and a commercially available kit (iScriptTM cDNA synthesis, Bio-Rad Catalog # 170-8891). Quantification of mRNA expression was performed similar to as previously described (Hindi and Kumar, 2016 (link); Fong et al., 2012 (link)) using the SYBR Green dye method on a 7300 Real-Time PCR system (Applied Biosystems) using gene-specific primers, detailed in Supplementary Table 1 .
Iscript cdna synthesis
Manufactured by Bio-Rad
Sourced in United States
The IScript cDNA Synthesis Kit is a tool for the reverse transcription of RNA into complementary DNA (cDNA). It provides the necessary reagents and protocols to efficiently convert RNA into cDNA for downstream applications, such as gene expression analysis and quantitative PCR.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (13 mentions)
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
Trizol, by Thermo Fisher Scientific (8 mentions)
Iq sybr green supermix, by Bio-Rad (7 mentions)
Itaq universal sybr green supermix, by Bio-Rad (5 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (5 mentions)
Direct zol rna miniprep kit, by Zymo Research (4 mentions)
Abi prism 7300 sequence detection system, by Thermo Fisher Scientific (4 mentions)
Turbo dnase, by Thermo Fisher Scientific (4 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Rnase free dnase set, by Qiagen (3 mentions)
Rneasy plus mini kit, by Qiagen (3 mentions)
Prism 7900ht real time pcr system, by Thermo Fisher Scientific (3 mentions)
Rneasy extraction method, by Qiagen (3 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (3 mentions)
Ssofast evagreen supermix, by Bio-Rad (3 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (3 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (2 mentions)
Itag universal syber green supermix, by Bio-Rad (2 mentions)
101 protocols using iscript cdna synthesis
1
Gastrocnemius Muscle RNA Extraction and qRT-PCR
2
Candeia Gene Expression Analysis
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Total RNA was used as template to synthesized cDNA with iScriptTM cDNA synthesis (Bio-Rad) according to the manufacturer’s instructions. After DNase treatment (Thermo Fisher Scientific), Q-PCR was performed using Power SYBR Green (Applied Biosystems) in a 10 μl reaction using the standard program of the CFX ConnectTM instrument (Bio-Rad). Data were analyzed using Bio-Rad CFX Manager version: 3.0.1215.0601 (Bio-Rad). The reference gene use for this study was obtained by homology to the Chrysolaena obovata elongation 1-α factor (KM597066). A Candeia elongation factor-encoding fragment was amplified from Candeia cDNA using primers designed on sequence of C. obovata which are conserved among asteraceae species (Supplementary Table 2 ). The product obtained was sequenced. Based on this sequence we designed a set of reference primers used in our study. All primers used are provided in the supporting information (Supplementary Table 2 ). Samples from Candeia trees were separated in branch (n = 5), leaf (n = 3), and root (n = 4).
3
Reverse Transcription of RNA to cDNA
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It was performed using the kit iScriptTM cDNA synthesis (Bio-Rad, cat. no 1708890). 500 ng of RNA were mixed with 2 µl of 5X iScript Reaction Mix and 0.5 µl of the enzyme iScript Reverse transcriptase in a volume of 10 µl. The thermal cycler (Axygen MaxyGeneTM II) was programmed as follows: Alignment for 5 min at 25 °C, reverse transcription for 20 min at 46 °C and inactivation for 1 min at 95 °C. The reaction was cooled to 4 °C and diluted to 5 ng/µl.
4
Quantitative Real-Time PCR Analysis
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The mRNA expression of validated genes was determined by real-time quantitative PCR. Brie y, undifferentiated and the Dex-differentiated CAD cells were cultured for 5 days before RNA extraction. RNA concentration and purity were evaluated using a Nanodrop TM 2000 spectrophotometer (Thermo Fisher Scienti c, USA). The cDNA was synthesized using iScript TM cDNA synthesis (catalog number 1708841, Bio-Rad, USA) according to the manufacturer's protocol. Quantitative real-time PCR of the validated genes was performed on an equal amount of cDNA from each condition using iTag Universal SYBER ® Green supermix (catalog number 1725121, Bio-Rad, USA) with the ABIPRISM-7500 sequence detection system and analysis software (Applied Biosystems, USA). The experiments were repeated in triplicate. The mRNA level of GAPDH was used as an internal control. Ct values of the sample were calculated, and transcript levels were analyzed by the 2 -∆∆Ct method. Primers used in this study are listed in Supplementary Table 1.
5
Quantitative RT-PCR Analysis of ECM22
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Total RNA was extracted from the transformant strains CEN.PK 102-5B ECM22 and from the wild-type strain, CEN.PK 102-5B WT, using the kit ZR Fungal/Bacterial RNA Miniprep (Zymoresearch/The epigenetics company). Strains were grown until exponential growth phase in minimal synthetic media with 2% w/v glucose. The retrotranscription to obtain cDNA was performed using the iScriptTM cDNA Synthesis (Bio-Rad Laboratories, Inc.) kit. The obtained cDNA was used to perform a reverse transcription quantitative PCR using SsoFastTM EvaGreen® Supermix with Low ROX (Bio-Rad Laboratories, Inc.) with the specific primers RtEcm_fw and RTEcm_rev (Table 3), according to the manufacturer's instructions. Expression levels of ECM22 were normalized to the expression of the Actin housekeeping gene. Relative expression was calculated using the DDCt method.
6
Quantitative Real-Time PCR Analysis
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The mRNA expression of validated genes was determined by real-time quantitative PCR. Brie y, undifferentiated and the Dex-differentiated CAD cells were cultured for 5 days before RNA extraction. RNA concentration and purity were evaluated using a Nanodrop TM 2000 spectrophotometer (Thermo Fisher Scienti c, USA). The cDNA was synthesized using iScript TM cDNA synthesis (catalog number 1708841, Bio-Rad, USA) according to the manufacturer's protocol. Quantitative real-time PCR of the validated genes was performed on an equal amount of cDNA from each condition using iTag Universal SYBER ® Green supermix (catalog number 1725121, Bio-Rad, USA) with the ABIPRISM-7500 sequence detection system and analysis software (Applied Biosystems, USA). The experiments were repeated in triplicate. The mRNA level of GAPDH was used as an internal control. Ct values of the sample were calculated, and transcript levels were analyzed by the 2 -∆∆Ct method. Primers used in this study are listed in Supplementary Table 1.
7
RNA Extraction and cDNA Synthesis
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The RNA extraction was performed for each body of larvae using illustra TM RNA spin Mini RNA Isolation kit (GE Healthcare) and the RNA quality was confirmed with 1% agarose gel. The concentration of total RNA was determined using Gen5™ software and equipment Synergy HT Multi-Mode Plate Reader (BioTek).
Total cDNA was synthesized from 200 ng of RNA using the iScriptTM cDNA Synthesis (Bio-Rad) kit and TGradient Thermocycler (Biometra). All assays were performed by following the kits' protocols.
Total cDNA was synthesized from 200 ng of RNA using the iScriptTM cDNA Synthesis (Bio-Rad) kit and TGradient Thermocycler (Biometra). All assays were performed by following the kits' protocols.
8
Gene Expression Analysis of Infected Cells
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500,000 GL261 cells were infected at an MOI of 1. Infected and uninfected cells (three parallel samples each) were harvested 16 h after infection. RNA was extracted using an RNeasy mini kit (QIAGEN, Germany). cDNA was synthesized from pooled RNA samples with iScript cDNA synthesis (Bio-Rad, Hercules, CA, USA) from 1 μg of extracted RNA. iQ SYBR Green supermix (Bio-Rad) and the primers listed in Table S1 . Each sample was run in duplicate, and the fold change of expression was quantified using the 2–ΔΔCt method.
9
Quantitative RNA Expression Analysis
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Total RNA was isolated from 1–2 × 106 cells dry pellets kept at −80 °C for less than a week using QIAgen RNeasy Plus Mini kit. cDNA was generated from RNA using iScript cDNA Synthesis (#1708891, BioRad) with random hexamers according to the manufacturer’s instructions. Real-time PCR reactions were performed using a Vii7 sequence detection system. Relative quantification of the genes was performed using Power SYBR Mix (2×) and specific primers for human c-MYC (Fwd: 5′-TCAAGAGGCGAACACACAAC-3′, Rev: 5′-GGCCTTTTCATTGTTTTCCA-3′), TGFβR1 (Fwd, 5′-GCACAACAAAATCACTATCCCATTAG-3′, Rev, 5′-CATTTGGAGCCAGAACACTGC-3′), SMAD3 (Fwd, 5′-CAGCTGTGTCTGCCAAACACA-3′, Rev, 5′-GGCCGGTGGTGTAATACTACCTG-3′), HOXA9 (Fwd, 5′-CAGACCCTGGAACTGGAGAA-3′, 5′-ATTTTCATCCTGCGGTTCTG-3′) and CDKN1A (Fwd, 5′-CCTCATCCCGTGTTCTCCTTT-3′, Rev, 5′-GTACCACCCAGCGGACAAGT-3′) and the 2−ΔΔCt method as described by the manufacturer using β-ACTIN (Fwd, 5′-AAACTGGAACGGTGAAGGTG-3′, Rev, 5′-AGAGAAGTGGGGTGGCTTTT-3′) to normalize data.
10
Quantitative Gene Expression Analysis
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RNA was isolated by Trizol isolation (Invitrogen) followed by iScript cDNA synthesis (Biorad) and amplification at 60°C using SYBR Green Supermix (Quantas) as previously done by us with primers against IL-10, TGFβ, CD33 and GAPDH11 (link). The relative gene expression was calculated by the ΔΔCt method where untreated cells were the experimental control and the housekeeping gene GAPDH was the internal control.
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