Zebrafish larvae were thawed, transferred to lysis buffer, and sonicated by pulsing 3–5 times with an Emerson Industrial Branson Sonifier® (Danbury, CT). RNA isolation was performed using 2-mercaptoethanol (MP Biomedicals) and a GeneJET RNA Purification Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. RNA quantity and quality were assessed using a BioDrop μLITE spectrophotometer (Cambridge, United Kingdom). Sample cDNA was prepared using an iScript reaction mix kit (Bio-Rad, Hercules, CA), diluted 1:9 with nuclease-free water, and stored at −80 °C until processing. Each RT-qPCR sample was prepared using 10 μL of 2× iQ SYBR® Green Supermix (Bio-Rad), 5 pmol (250 nM) each of forward and reverse primers (1 μL total), 5 μL of nuclease-free water, and 4 μL (10 ng) of cDNA. Samples were run on 96-well plates in a CFX Connect Real-Time PCR Detection System (Bio-Rad), and samples were analyzed using the CFX Manager software (Bio-Rad). RT-qPCR was carried out in duplicate for the cyp1a and ahr2 genes. The β-actin (actb) gene was used as a housekeeping gene, and its transcription did not change significantly across exposure groups.
Iscript reaction mix kit
Manufactured by Bio-Rad
Sourced in Germany, United States
The IScript reaction mix kit is a ready-to-use solution for reverse transcription and real-time PCR. It contains all the necessary components for cDNA synthesis and subsequent qPCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Cfx manager software, by Bio-Rad (3 mentions)
Rneasy plus universal kit, by Qiagen (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Aurum total rna fatty and fibrous tissue kit, by Bio-Rad (2 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
μlite spectrophotometer, by Harvard Bioscience (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
2 mercaptoethanol, by MP Biomedicals (1 mentions)
Dynabeads mrna direct kit, by Thermo Fisher Scientific (1 mentions)
Ultra turrax disperser, by IKA Group (1 mentions)
5 protocols using iscript reaction mix kit
1
Zebrafish RNA Extraction and RT-qPCR Analysis
2
Quantitative RT-PCR Analysis of Colon Macrophages
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Pieces of frozen tissue were homogenized and total RNA extracted with the help of the RNeasy plus Universal Kit (Qiagen, Hilden, Germany). To prevent inhibition of the reverse transcriptase by residual DSS present in the RNA preparation [25 (link), 26 (link)], mRNA was purified using the Dynabeads mRNA DIRECT Kit (ThermoFisher). Thereafter, the mRNA was reverse transcribed with a iScript Reaction Mix kit (Bio-Rad, Munich, Germany) followed by quantitative RT-PCR using the Power SYBR Mix (ThermoFisher). Results were normalized to the expression of the house-keeping gene HPRT and evaluated using the ΔΔCt method. In order to correct the mRNA levels of each gene for differences in macrophage numbers in the colon, the obtained values were finally normalized to F4/80 gene expression.
3
RNA Extraction and RT-qPCR Analysis
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Frozen tissue was homogenized using an Ultra-Turrax disperser (IKA-Werke, Germany) and RNA extraction was performed with the help of the RNeasy plus Universal Kit (Qiagen, Hilden, Germany) according to the manufacturer´s instructions. 1 μg RNA was reverse transcribed with the iScript™ Reaction Mix kit (Bio-Rad, Munich, Germany) and quantitative RT-PCR (RT-QPCR) was performed using the Power SYBR® Mix (Applied Biosystems, Darmstadt, Germany). Results were normalized to the expression of the house-keeping gene HPRT and evaluated using the ΔΔCt method. Primer sequences are available upon request.
4
RNA Extraction and RT-qPCR Analysis
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The total RNA was isolated from SAF-1 cells using the RNA extraction kit (Aurum Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad, Hercules, CA, USA). Then, 1 μg of the total RNA was converted into cDNA using the 5X iScript Reaction Mix Kit (Bio-Rad, Hercules, CA, USA). RT-qPCR was performed in a 20 μL reaction system using the 1X IQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). The relative mRNA level of a target gene was quantified using the comparative cycle threshold (2−ΔΔCT) method [105 (link)]. The 18s of the endogenous reference and the relative quantification of (pparβ, pparγ, D6D, fas, cd36, fatp1, and fabp1) gene expression was evaluated after normalization with the reference genes. Data processing and statistical analyses were performed using the CFX Manager Software (Bio-Rad, Hercules, CA, USA). The primers used are shown in Table 6 .
5
Transcriptional Profiling of Microalgae
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Total RNA was extracted from fresh samples at time zero and after 15 days of monitoring in nitrogen standard and stress cultures. RNA was isolated from samples in PureZOL™ using the Aurum Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad, Hercules, CA, USA) and was measured spectrophotometrically using a Thermo Scientific™ µDrop Plate Spectrophotometer. All samples were analyzed in triplicate. Then, reverse transcription was performed using the 5X iScript Reaction Mix Kit (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions.
The amplification and the relative quantification were performed in triplicate on genes G6PDH, rbcL, FCP B, ACCase, Cit syn, and isocit DH (Table 1 ). Relative gene expression was evaluated after normalization with the reference genes. Data processing and statistical analysis were performed using CFX Manager Software (Bio-Rad, Hercules, CA, USA). The relative expression of all genes was calculated by the 2−ΔΔCT method [34 (link)] using P. tricornutum RPS (ribosomal protein S1) and TBP (TATA box-binding protein) as the endogenous reference.
The amplification and the relative quantification were performed in triplicate on genes G6PDH, rbcL, FCP B, ACCase, Cit syn, and isocit DH (
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