Total RNA was isolated from primary human skin fibroblasts, human ESCs (HUES-1, -2, -4), Embryoid Bodies (EBs), ESC-derived monocytes, human iPSCs, iPSC-derived motor and -dopaminergic neurons from SMA and Parkinson'disease patients, respectively, using either Trizol reagent (Invitrogen) or Qiagen's (mi)RNA purification kits according to the manufacturers’ instructions. Total RNA extracted from human tissues, including fetal and adult brain, lung, spleen (fetal only), kidney, heart and placenta (adult only), was obtained from Agilent technologies. cDNA was generated from these samples using Superscript III (Invitrogen), according to manufacturers’ instructions. Real-time qPCR was performed using a QuantiTect SYBER Green mastermix (Qiagen). All oligonucleotides used in qPCR reactions are outlined in Supplementary Table S2.
Quantitect syber green master mix
Manufactured by Qiagen
Sourced in Germany
QuantiTect SYBR Green PCR Master Mix is a ready-to-use solution for real-time quantitative PCR (qPCR) using SYBR Green I dye. It contains all the necessary components, including HotStarTaq DNA Polymerase, SYBR Green dye, dNTPs, and optimized buffer system, to perform reliable and sensitive qPCR.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Mircury rna isolation kit, by Qiagen (2 mentions)
Taqman universal mix, by Thermo Fisher Scientific (2 mentions)
Miscript pcr kit, by Qiagen (2 mentions)
Expression suite, by Thermo Fisher Scientific (2 mentions)
Qubit 2, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Quantstudio 5, by Thermo Fisher Scientific (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Rotor gene pcr analyzer, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Quantitect rt kit, by Qiagen (1 mentions)
7 protocols using quantitect syber green master mix
1
Comprehensive RNA Isolation and qPCR Analysis
2
Quantitative Real-Time PCR Protocol
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qRT–PCR was performed on 5 ng of cDNA using a final concentration of 2 μM each of forward and reverse primers (see primers list) using Quantitect Syber Green Master Mix (Qiagen) on an Applied Biosystems StepOne Plus or QuantStudio5 real-time PCR system. Experiments were analysed using the ΔΔCt method. Statistical analysis was performed using Prism 7 software. Samples were normalized relative to the expression of an endogenous control gene (Rplp0; see primers list for sequences). Amplification primers were designed using Primer 3.1 to span one intron and purchased from Sigma-Aldrich, and are listed in Supplementary Tables 4 and 5 .
3
Quantitative Analysis of Apoptosis Genes
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A concentration of 500 ng of freshly extracted mRNA at 48 h, was immediately used for cDNA synthesis by T-100 thermal cycler (Bio-Rad, CA, USA). The high-capacity cDNA reverse transcriptase Kit (Thermo Fisher Scientific, MA, USA) was used following the manufacturer’s instructions and then cDNA samples were immediately kept at − 20°C till further PCR amplification.
The obtained cDNA was subsequently amplified using Quantitect Syber green Master mix (Qiagen, Hilden, Germany), by StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fischer Scientific, USA). The PCR program was adjusted as follows: enzyme activation step, 10 min at 95 °C. The amplification step, 40 cycles of 15 s at 95 °C, 20 s at 55 °C, and 30 s at 72 °C. The following primer sequences of apoptosis-related genes and β-actin (Ramadan et al. 2019 (link)) were used as follows:
The obtained cDNA was subsequently amplified using Quantitect Syber green Master mix (Qiagen, Hilden, Germany), by StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fischer Scientific, USA). The PCR program was adjusted as follows: enzyme activation step, 10 min at 95 °C. The amplification step, 40 cycles of 15 s at 95 °C, 20 s at 55 °C, and 30 s at 72 °C. The following primer sequences of apoptosis-related genes and β-actin (Ramadan et al. 2019 (link)) were used as follows:
BAX: F 5'-ATG GAC GGG TCC GGG GAG CA -3', R 5'- CCC AGT TGA AGT TGC CGT CA-3'
BCL-2: F 5'- GTG AAC TGG GGG AGG ATT GT -3', R 5'- GGA GAA ATC AAA CAG AGG CC -3'
TP53: F 5'- TCA GAT CCT AGC GTC GAG CCC-3', R 5'- GGG TGT GGA ATC AAC CCA CAG-3'
Β-actin: F 5’- CTG TCT GGC GGC ACC ACC AT-3’, R 5’- GCA ACT AAG TCA TAG TCC GC-3’.
4
Quantitative Gene Expression Analysis
5
Quantitative Analysis of NF-κB Gene Expression
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After 14 days of incubation in Osteogenic media, extraction of a total of mRNA was achieved using the RNeasy Mini Kit (Qiagen, Hildesheim, Germany). in accordance with the manufacturer's instructions, using the Quantitect RT Kit (Qiagen, Hilden, Germany) Table 1 . “Quantification” of NF-k gene expression was amplified from total RNA extracts using Quantitect primer assay - primer assays, cat no: 249900; [Hs_NF-k, ID: QT00396823]. Primordial Test. Quantitect Syber green Master mix served to amplify the genes (Qiagen, Hilden, Germany). Similarly in earlier research, the -actin (Hs ACTB) primer assay (ID: QT000954231) was utilized as a housekeeping gene (Abdelgawad et al., 2021 (link)). The five-plex Rotor-Gene PCR Analyzer was used to evaluate all samples (Qiagen, Germany). Gene expression levels were analyzed using the ΔCt technique, with B-actin serving as a housekeeper gene for normalization. The tests were repeated in triplicate.
6
Quantitative Gene Expression Analysis
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Cells were plated the day before the experiments onto 6-well plates (3x105) or 12-well plates (1x105). Total RNA was isolated using RNeasy Kit (Qiagen). miRCURY™ RNA Isolation Kit (Exiqon, Denmark) was used for microRNAs extraction. RNA isolation was carried following manufacturer’s protocols. RNA was quantified using the fluorimeter Qubit 2.0 (Life Technologies) following manufacturer’s instructions or Nanodrop (Thermo). Reverse transcription of RNA was performed using Quantitect-Reverse transcription kit (Qiagen) or miScript PCR kit (Qiagen) using 300-500 ng of total RNA. Real time qPCR was performed using Quantitect Syber Green master mix (Qiagen) or Taqman universal mix (Life Technology) on a Step One Plus real-time PCR system (Life Technology). Experiments were analysed using the software Expression Suite (Life Technology) and StepOne software 2.3 and Relative quantification (RQ) with max and min values (RQ max and RQ min) were calculated using S.D. algorithm. Statistical analysis was performed using Expression Suite software on at least three independent cultures. Housekeeping genes used for internal normalisation are β-Actin for mRNA and Snord95 Snord61 and RNU6B, for miRNAs. The primers were designed using ProbeFinder- Roche or purchased by Qiagen and are listed in SI Table 1 .
7
Quantifying Microbial Functional Genes
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Frozen 20-ml FTW water samples were thawed and centrifuged for 5 min at 2,500 × g to pellet bacterial biomass. The supernatant was carefully decanted, and the pellet was resuspended in sterile distilled water and transferred to 2ml vials. Following the protocol of Ausubel et al. (1989) , vials were boiled to lyse microbial cells and then centrifuged at 10,000 × g for 10 min at 2 ˚C before transferring the clarified supernatant containing DNA to a clean tube. Six genes, including 16S ribosomal RNA (Muyzer et al., 1993) (link), archaeal and bacterial ammonia monooxygenase (amoA) from nitrifying microorganisms (Tourna et al., 2008; (link)Rotthauwe et al., 1997) , and two nitrate reductases (nirS and nirK) and a nitrous oxide reductase (nosZ) from denitrifying bacteria (Braker et al., 1998) (link), were quantified in DNA extracts using quantitative polymerase chain reaction (PCR). Primer descriptions and PCR procedures can be found in Supplemental Table S2. Quantitative PCR reactions were carried out with QuantiTect Syber Green master mix (QIAGEN) using a StepOnePlus real-time PCR system (Applied Biosystems Inc.). Each sample was measured in triplicate, averaged, converted to copies per milliliter of original sample, and log transformed. Archaeal amoA and bacterial amoA, nirS, nirK, and nosZ gene abundances were analyzed relative to 16S concentration.
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