Total RNA was extracted using RNeasy Mini kit (QIAGEN, Germantown, MD, USA). First-strand complementary DNA was synthesized with iScript cDNA synthesis kit according to manufacturer's protocol (Bio-Rad 170-8890, Hercules, CA, USA). An equal volume mixture of the products was used as templates for PCR amplification. Reactions were performed in a 25 μl volume with iQ SYBR Green Supermix (Bio-Rad) and 200 nM each of forward and reverse primers shown in Supplementary Table 3 using iCyler and iQ software (Bio-Rad). Each sample was run in duplicate. PCR conditions included an initial denaturation step of 4 min at 95 °C, followed by 40 cycles of PCR consisting of 30 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. Average threshold cycle values from the duplicate PCR reactions for a gene of interest were normalized against the average threshold cycle values for GAPDH from the same complementary DNA sample.
Icyler
Manufactured by Bio-Rad
Sourced in United States
The iCycler is a real-time PCR detection system designed for quantitative gene expression analysis. It features a thermal cycler and sensitive fluorescence detection capabilities to monitor amplification of DNA samples in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Iq software, by Bio-Rad (3 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Iqtm sybr green supermix, by Bio-Rad (2 mentions)
Gotaq flexi buffer, by Promega (1 mentions)
Mgcl2, by Promega (1 mentions)
Dntps, by Promega (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
6 protocols using icyler
1
Quantitative Real-Time PCR Protocol
2
Quantitative Real-Time PCR Analysis
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Real‐time quantitative PCR (qRT‐PCR) was performed on total RNA isolated from induced neurons at different time points as described previously (Livak & Schmittgen, 2011 ). First‐strand complementary DNA (cDNA) was synthesized using SuperScript First‐Strand Synthesis System (Life Technologies, Invitrogen). For each sample, 5 μl cDNA product was used as the template for PCR amplification in a 25 μl reaction volume containing iQTM SYBR Green Supermix (Bio‐Rad) and 200 nM of each primer. The reaction was conducted using iCyler and iQ software (Bio‐Rad). The PCR conditions included an initial denaturation step of 4 min at 95°C, followed by 40 cycles of PCR consisting of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. Each reaction was carried out in triplicate independently. The average threshold cycle (Ct) values from the triplicate PCR reactions for a gene of interest (GOI) were normalized against those for GAPDH from the same cDNA sample. The fold change of GOI transcript levels between sample X and sample Y is calculated as 2−∆∆Ct, where ∆Ct = Ct (GOI) − Ct (GAPDH), and ∆∆Ct = ∆Ct (X) − ∆Ct (Y) (Livak & Schmittgen, 2011 ).
3
Quantitative Real-Time PCR for Gene Expression
4
Quantitative Analysis of Stem Cell Gene Expression
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Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed on total RNA isolated from naivetropic iPSCs grown on gelatin or midbrain neurons differentiated from naivetropic iPSCs. First-strand complementary DNA (cDNA) was synthesized by SuperScript First-Strand Synthesis System (Life Technologies). For each sample, 5 μL cDNA product was used as the template for polymerase chain reaction (PCR) amplification. Reactions were performed in a 25 μL volume with iQ™ SYBR Green Supermix (Bio-Rad) and 200 nM each primers shown in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/scd ) using iCyler and iQ software (Bio-Rad). Each sample was run in triplicate. Average threshold cycle (Ct) values from the triplicate PCR reactions for a gene of interest (GOI) were normalized against the average Ct values for GAPDH from the same cDNA sample. Fold change of GOI transcript levels between sample X and sample Y equals 2−ΔΔCt, where ΔCt = Ct(GOI) −Ct(GAPDH), and ΔΔCt = ΔCt(X) −ΔCt(Y).
5
Transcriptome-Derived SSR Primers for Onobrychis
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A total of 400 SSR primers designed from O. viciifolia transcriptome data (Mora-Ortiz 2016, unpublished), were tested with unlabeled primers for amplification in the 32 plants using an iCyler (Biorad, Hercules, USA) in a volume of 10 μL, with 10 ng DNA, 1 x Go Taqflexi buffer (Promega, Madison, USA), 2.5 mM MgCl2 (Promega), 0.2 mM dNTPs (Promega), 0.2 μM forward primer, 0.2 μM reverse primer and 0.5 U Polymerase G2 (Promega). The conditions followed a touchdown PCR approach with 4 min at 94 °C, 12 cycles of 30 s at 66 °C with −1 °C decrease at each cycle plus 30 s at 72 °C, and 30 cycles of 30 s at 94 °C, 30 s at 54 °C plus 30 s at 72 °C, followed by 7 min at 72 °C. PCR products were separated by gel electrophoresis. Amplicons were separated on 1 % agarose in 1x TBE buffer, stained with ethidium bromide and visualized under UV light.
6
RNA Isolation from M. tuberculosis
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For RNA isolation from in vitro bacterial cultures, M. tuberculosis strain Erdman was passaged three times in SMT, then replicate cultures were grown in SMT inoculated to OD600 = 0.05. At OD600 = 0.35, TTM was added to 20 µM to the half of the cultures. At OD600 = 0.45, bacteria were harvested by centrifugation (10,000× g, 5 min) and the pellets stored at −80 °C. RNA was isolated as described previously [16 (link)]. For RNA isolation from the M. tuberculosis-infected 3T3-L1 adipocytes, host cells infected at MOI = 1 were cultured in basal medium (DMEM-FBS 10%) alone or with addition of either 0.1 mM CuCl2 or ZnSO4 and incubated at 37 °C, 5% CO2 with medium replaced every 3 days. At day 10 post-infection, cells were lysed by suspension in 4 M guanidine isothiocyanate and bacteria harvested by centrifugation (10,000× g, 45 min). Bacterial RNA was isolated from the pellets using the methods described earlier for isolation from axenic cultures. For cDNA synthesis, 0.5 µg RNA was used with 200 ng random hexamers and Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer instructions. Quantitative PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems), gene-specific primers, and prepared cDNA on a BioRad iCyler. For ΔΔCt analysis, gene expression was normalized to the M. tuberculosis housekeeping gene sigA [16 (link)].
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