After extraction, ribonucleic acid (RNA) was reverse-transcribed with RevertAid H minus Reverse transcriptase kit (Thermo Fisher Scientific, UK) with 0.3 µg of random hexamer, 20 U of RNase inhibitor, and 1 mM of each dNTP following the manufacturer's protocol in a thermocycler (Applied Biosystems). For the expression measurement, the reaction mix contained 2.5 µL of complementary desoxyribonucleic acid diluted 10-fold, 200 nM of each primer (Table S1), and 2x iTaq UniverSybr Green® Supermix (Bio-Rad) in a final volume of 20 µL. The cycling conditions were 95°C for 3 min followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. To control the specificity of the amplification products, a melting curve analysis was performed. Copy number was calculated from the standard curve. Expression levels of target genes were normalised to the geometric mean of the values for the housekeeping genes (ribosomal protein S13, S18 and L27 in human lung tissue and primary HBEC, S18 and hypoxanthine-guanine phosphoribosyltransferase 1 in Calu-3, and TATA-box binding protein, hypoxanthine-guanine phosphoribosyltransferase 1 and tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein zeta in mouse tissue).
Itaq univer sybr green supermix
Manufactured by Bio-Rad
ITaq Univer SYBR Green Supermix is a ready-to-use PCR master mix that contains the necessary components for real-time quantitative PCR amplification and detection using SYBR Green I dye.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (4 mentions)
Thermocycler, by Thermo Fisher Scientific (3 mentions)
Revertaid h minus reverse transcriptase kit, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Cfx software, by Bio-Rad (2 mentions)
Iscript rt supermix, by Bio-Rad (1 mentions)
Mn nucleospin rna kit, by Macherey-Nagel (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Primer express software 3.0 for rt pcr, by Thermo Fisher Scientific (1 mentions)
Aurum total rna mini kit, by Bio-Rad (1 mentions)
Cfx96 touch detection system, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
8 protocols using itaq univer sybr green supermix
1
Reverse Transcription and qPCR Analysis
2
Smad4 Expression in Treated NK Cells
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Hippurate, 3-hydroxyphenyl-acetic acid, and 2,4,6-trihydroxybenzoic acid monohydrate treated NK cells (2x105 cells per dose, duplicate) were isolated to determine Smad4 gene expression, Total mRNA was reverse-transcribed using iScript RT Supermix (Bio-Rad, Hercules, CA, USA). PCR was performed as described previously (40 (link)–42 (link)) with iTaq Univer SYBR Green Supermix (Bio-Rad). Human Smad4 primers were purchased from Integrated DNA Technologies (IDT), Inc. (Coralville, IA, USA). Relative expression of a gene in cells was determined by comparing the threshold cycle (Ct) of the gene against the Ct of a housekeeping gene, Gapdh.
3
Gene Expression Analysis by qPCR
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For every experiment, cells were cultured in triplicates for each condition and total RNA extraction was performed using MN NucleoSpin RNA Kit (Macherey-Nagel). Experiments were then independently repeated. RNA was quantified and reverse transcribed to cDNA using High-capacity cDNA reverse transcription kit (Applied Biosystem) to a final concentration of 2.5 ng/μl. Quantitative real-time PCR (QPCR) was performed using CFX384 with iTaqUniver SYBR Green Supermix (Biorad). QPCR assays were performed in duplicate for each sample, the quantification of each gene expression was normalized to ACTIN, calculated by 2−ddCt method. p < 0.05 (∗) indicates statistical significance by ANOVA and Dunnett’s post-hoc tests. All error bars indicate standard error of mean of biological triplicates. Primers used are listed in Table S8 .
4
Quantitative PCR Analysis of Gene Expression
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RNA extraction, reverse transcription, and RT-qPCR were performed as previously described [43] (link). Total RNA was extracted from reconstituted ALI-cultured epithelia using TRIzol reagent (Thermo Fisher Scientific). 500 ng of RNA was reverse-transcribed with RevertAid H Minus Reverse transcriptase kit with 0• 3 µg of random hexamer, 20 U of RNase inhibitor and 1 mM of each dNTP (Thermo Fisher Scientific) following the manufacturer's protocol in a thermocycler (Applied Biosystems, Foster City, CA). The expression levels were quantified by real-time quantitative PCR using the CFX96 PCR (Bio-Rad, Hercules, CA). The reaction mix contained 2• 5 µl of complementary desoxyribonucleic acid diluted 10-fold, 200 nM of each primer (primers properties are detailed in Table S3) and 2x iTaq UniverSybr Green® Supermix (Bio-Rad) in a final volume of 20 µl. The cycling conditions were 95 °C for 3 min followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. To control the specificity of the amplification products, a melting curve analysis was performed. The copy number was calculated from the standard curve. Data analysis was performed using Bio-Rad CFX software (Bio-Rad). Expression levels of target genes were normalized to the geometric mean of the values of the 3 housekeeping genes (RPL27, RPS13, RPS18).
5
Quantitative RNA Expression Analysis Protocol
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RNA extraction, reverse transcription, and qRT-PCR were performed as previously described (Bustin et al, 2013 (link)). Total RNA was extracted from reconstituted ALI-cultured epithelia using TRIzol reagent (Thermo Fisher Scientific). 500 ng of RNA was reverse-transcribed with RevertAid H minus reverse transcriptase kit with 0.3 μg of random hexamer, 20 U of RNase inhibitor, and 1 mM of each dNTP (Thermo Fisher Scientific) following the manufacturer’s protocol in a thermocycler (Applied Biosystems). The expression levels were quantified by real-time quantitative PCR with the CFX96 PCR (Bio-Rad). The reaction mix contained 2.5 μl of complementary desoxyribonucleic acid diluted 10-fold, 200 nM of each primer (primers properties are detailed in Table S2), and 2x iTaq UniverSybr Green Supermix (Bio-Rad) in a final volume of 20 μl. The cycling conditions were 95°C for 3 min followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. To control the specificity of the amplification products, a melting curve analysis was performed. The copy number was calculated from the standard curve. Data analysis was performed using Bio-Rad CFX software (Bio-Rad). Expression levels of target genes were normalized to the geometric mean of the values of three housekeeping genes (RPL27, RPS13, RPS18).
Table S2
6
Quantitative Analysis of Gene Expression
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Total RNA was extracted from cells and tissues using TRIzol reagent (Invitrogen). Then, the RNA was reversetranscribed into complementary DNA (cDNA) using a PrimeScript RT reagent kit (Takara, RR037A). RT-qPCR analysis was conducted using iTaq Univer SYBR Green Supermix (Bio-Rad) and QuantStudio5 Applied Biosystems (Thermo Fisher Scientific) according to the manufacturer's instructions. The results were analyzed as the fold change in expression. The primer pairs used are listed below:
7
Quantitative Real-Time PCR Analysis
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Total RNA from cultured cells of desired genotypes was extracted with TRIzol reagent (Thermo, 15596026) and genomic DNA was removed using DNA-freeTM Kit DNase Treatment and Removal Reagents (Thermo, AM1906) according to the manufacturer’s protocol. Total RNA was then converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo, 4368814). Quantitative real-time PCR reactions were performed according to the protocol of the iTaq Univer SYBR Green Supermix (Bio-Rad, 1725125). The same PCR program was used for all quantifications: 95 °C for 3 min, 95 °C for 10 s, 55 °C for 10 s, and 72 °C for 30 s. This cycle was repeated 40 times, followed by melting curve measurement. The sequence of primers used for mRNA are list in Supplementary Table 1 . Primers of the actin gene were designed as a real-time PCR control. ΔΔCt method was used for normalized expression. Data analysis was performed in accordance with MIQE guidelines70 (link).
8
Quantitative mRNA Expression Analysis
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