FOXO3a expression was validated by an SYBR green-based quantitative real-time polymerase chain reaction (qRT-PCR) assay (Qiagen, Valencia, USA). Primers for FOXO3a and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Invitrogen and are listed in Table 2 . The qRT-PCR was performed in an ABI7900 cycler (Applied Biosystems, Foster City, USA), and the cycle threshold (Ct) value was recorded.
Abi 7900 cycler
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 7900 cycler is a real-time PCR instrument designed for high-throughput gene expression analysis and genetic variation detection. It features a 384-well format and supports a wide range of quantitative PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Purelink kit, by Thermo Fisher Scientific (3 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Quanta cdna supermix, by Quanta Biosciences (2 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Ifnα2a, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green rt pcr kit, by Qiagen (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Glass bead, by Merck Group (1 mentions)
Superscript 4 vilo, by Thermo Fisher Scientific (1 mentions)
Acid phenol chloroform isoamyl alcohol, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dna free kit, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Chloroform, by Merck Group (1 mentions)
Rnaiso, by Takara Bio (1 mentions)
Primescript first strand cdna synthesis kit, by Takara Bio (1 mentions)
Mirna reverse transcription kit, by RiboBio (1 mentions)
9 protocols using abi 7900 cycler
1
Quantifying FOXO3a Expression by qRT-PCR
2
Quantifying IFN-induced gene expression
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For IFN stimulation, 7.5 × 105 cells/12-well (RNA samples for quantitative real-time reverse transcription PCR (RT-qPCR)) or > 5 × 105 cells/12-well (whole-cell lysates for immunoblotting) were seeded. MDMs were left untreated or treated with 1000 U/mL IFNα2a (PeproTech), IFNβ1a (PBL Interferon Source) or IFNγ (PeproTech) for 8/24 h (RNA samples) or 24 h (whole-cell lysates) at 37 °C. As a control for IFN induction, MDMs were infected with Sendai virus (final dilution = 1:200).
Total RNA from MDMs was isolated using the RNeasy Plus Mini Kit (QIAGEN) or NucleoSpin RNA Kit (Macherey-Nagel). Expression of mRNAs was determined in a 384-well format using QuantiTect SYBR Green RT-PCR Kit (QIAGEN) on an ABI7900 cycler (Applied Biosystems). Isoform-specific primers are listed in the Supplementary Methods.
Total RNA from MDMs was isolated using the RNeasy Plus Mini Kit (QIAGEN) or NucleoSpin RNA Kit (Macherey-Nagel). Expression of mRNAs was determined in a 384-well format using QuantiTect SYBR Green RT-PCR Kit (QIAGEN) on an ABI7900 cycler (Applied Biosystems). Isoform-specific primers are listed in the Supplementary Methods.
3
Quantitative PCR Analysis of unc-51 in C. elegans
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RNA was isolated from young adult animals. Worms were homogenized, and RNA was extracted with Trizol reagent (Invitrogen) using repeated cycles of vortexing (4 °C) with 500- µm glass beads (Sigma) and freeze-thawing in liquid N2. RNA was isolated with chloroform (Sigma) and acid phenol:chloroform:Isoamyl alcohol (125:24:1), pH 4.5 (Ambion). Contaminating DNA was removed by subjecting 10 µg of RNA to DNA-free Kit (Ambion). cDNA was generated from 1 µg of RNA with Superscript IV Vilo (Invitrogen). Quantitative PCR was carried out using Power-Up Sybr Green Master Mix in the ABI 7900 cycler (Applied Biosystems). All qPCR amplifications were performed in triplicate using cDNA and no RT negative controls. Quantitation of unc-51 was normalized to tba-1, pmp-3, and Y45F10D.4 reference genes75 (link) using the comparative C(T) method for analysis76 (link). All primers used for qPCR are listed in Table S5 .
4
Quantitative Real-Time PCR Protocol
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Total RNA was prepared using the Purelink Kit (Ambion). Total RNA was further reverse-transcribed to cDNA with SuperScript III Reverse Transcriptase (Invitrogen) and analyzed by quantitative real-time PCR on the Applied Biosystems SYBR Green PCR Master Mix with an ABI-7900 cycler (Applied Biosystems). List of primers used and sequences is provided in Supplemental Table 1 .
5
Quantification of Transcripts in Mouse Brain
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RNA was isolated from the tissues dissected from the adult mouse brain as described earlier and cDNA was generated by reverse transcription as described earlier (Kadakkuzha et al., 2013 (link)). Briefly, 1 μg of RNA was used with Quanta cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD) according to the manufacturer's instructions and the expression of transcripts were quantified by qRT-PCR using SYBR Green PCR master mix (Applied Biosystems Carlsbad, CA) for detection in ABI 7900 cycler (Applied Biosystems Carlsbad, CA). All the qPCR amplifications were performed in quadruplicate in a total volume of 10 μl containing 2 μl of H2O, 2 μl of cDNA, 5 μl of 2X Master Mix, 1.0 μl of 10 μM (each) forward and reverse primers. Quantification of each transcript was normalized to the mouse 18S reference gene following the 2−ΔΔCt method (Livak and Schmittgen, 2001 (link)). Student t-test was used to select genes with statistically significant expression levels where *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001. The sequences of primers for the mRNAs and lncRNAs are given in the Table S3 .
6
Quantification of miRNA and mRNA Levels
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Both tissue samples and cultured cells were used to extract total RNA using RNAiso (TaKaRa, Japan), following manufacturer’s protocols. Next, 1 µg of the RNA was reverse-transcribed into cDNA with miRNA Reverse Transcription Kit (RiboBio, China) and miR-33b levels were measured using the miRNA kit (RiboBio, China) and normalized using the endogenous U6 control. To verify our findings and to examine alterations in the transcription of relevant genes, 2 µg of total RNA was reverse transcribed using a PrimeScript first-strand cDNA synthesis kit (Takara, Japan) and qPCR was executed using SYBR® Premix Ex Taq™ II (Takara, Japan) following manufacturer’s guidelines. Supplementary Table 1 lists the primers used in this study. ABI 7900 cycler (Applied Biosystems, Foster City, CA) was employed for the qPCR amplification and GAPDH was employed as the endogenous control. The relative expression of relevant genes were determined with the 2−ΔΔCt method.
7
Total mRNA Extraction and qPCR Analysis
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Purelink Kit (Ambion, Life technologies, Carlsbad, CA) was used to prepare total mRNA, which was further reverse-transcribed to cDNA with SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA). Quantitative real-time PCR (qPCR) analysis was performed using SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA) with an ABI-7900 cycler (Applied Biosystems).
8
Quantitative Analysis of Transcript Expression
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The RNA from the LCM samples was reverse transcribed to cDNA using the same method previously reported from this laboratory (Kadakkuzha et al., 2013 (link); Raveendra et al., 2018 (link)). 1 μg of RNA was used with Quanta cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD) according to the manufacturer’s instructions and the expression of transcripts were quantified by qRT-PCR using SYBR Green PCR master mix (Applied Biosystems Carlsbad, CA) for detection in ABI 7900 cycler (Applied Biosystems Carlsbad, CA). Quantification of each transcript was normalized to the mouse 18S reference gene following the 2−ΔΔCt method (Livak and Schmittgen, 2001 (link); Kadakkuzha et al., 2013 (link)). One-way ANOVA and Student-Newman test was used to select genes with statistically significant expression levels.
9
Quantitative real-time PCR analysis
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Total RNA was prepared using the Purelink Kit (Ambion, Life technologies). Total RNA was further reverse-transcribed to cDNA with SuperScript III Reverse Transcriptase (Invitrogen) and analyzed by quantitative real-time PCR using ABI-7900 cycler (Applied Biosystems). The genes were normalized to Ppia for the tissues or Hprt for the myotubes. List of primers used and sequences is provided (S2 Table ).
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