RNA was extracted from ~15 mg of biopsied muscle using the RNeasy kit (QIAGEN 74104) after a 60-s homogenization in QIAzol Lysis Reagent (QIAGEN, Hilden, Germany, 79306). One microgram of RNA in a total volume of 20 μl was reverse-transcribed to cDNA using iScript cDNA Synthesis Kit (#170–8890) (Bio-Rad, Hercules, CA, USA). Real-time PCR was performed using CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), PrimePCR™ Probe Assay (Bio-Rad, Hercules, CA, USA) and iQ Supermix kit (#170–8862) (Bio-Rad, Hercules, CA, USA). The cycling parameters were: 95°C for 3 min, then 50 cycles at 95°C for 10 s and 58°C for 30 s. Gene expression, normalized to the geometric mean of a housekeeping genes (RPP30), was quantified using the 2−(ΔCt) method and expressed as fold difference relative to the RPP30. The primers and probes used to amplify target were supplied from Bio-Rad PrimePCR™ Probe Assay: p16INK4a (or CDKN2A) (Assay ID: qHsaCEP0057827), CD34 (Assay ID: qHsaCIP0026476), GLB1 (Assay ID: qHsaCEP0057625), MPO (Assay ID: qHsaCEP0049167), and RPP30 (Assay ID: qHsaCEP0052683). Test-retest reliability values for p16INK4a mRNA and MPO mRNA are r = 0.87 and r = 0.94, respectively.
Iq supermix kit
Manufactured by Bio-Rad
Sourced in United States
The IQ Supermix kit is a real-time PCR reagent designed for sensitive and specific detection of target DNA sequences. It contains all necessary components, including a hot-start polymerase, buffers, and dNTPs, to perform quantitative PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Taqman mirna assay kit, by Thermo Fisher Scientific (3 mentions)
Iscript select cdna synthesis kit, by Bio-Rad (3 mentions)
Iq sybr green mix, by Bio-Rad (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Pcr flatform, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Primepcr probe assay, by Bio-Rad (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Nucleospin rna 2 kit, by Macherey-Nagel (1 mentions)
Trizol extraction, by Thermo Fisher Scientific (1 mentions)
C1000 thermocycler, by Bio-Rad (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Geneamp gold rna pcr reagent kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using iq supermix kit
1
Quantifying mRNA Expression in Muscle Biopsies
2
Quantitative RT-PCR for Mouse and Human Genes
3
Quantifying proviral mRNA transcripts
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For the mRNA analysis of the proviral transcripts, total RNA was isolated from cells using Trizol extraction (ThermoFisher Scientific, Grand Island, NY, USA) according to the manufacturer’s instructions. Five hundred nanograms of total RNA was used to generate cDNA with the iQ Supermix kit (Bio-Rad) using oligo-dT reverse primers. The full length and short abortive proviral transcripts were then quantified by qRT-PCR using envelope (ENV) and TAR responsive gene primers, respectively, that have been previously described [59 (link)]. The qRT-PCR assays were performed using the C1000 Thermo Cycler with the CFX96 Real-Time System and iTaq Universal SYBR Green Supermix (Bio-Rad). Cycling conditions were as follows: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 3 min, and 44 cycles at 95 °C for 15 s and 60 °C for 40 s. The absolute quantification was calculated based on the threshold cycle (Ct) relative to the standard curve and then the copy number was normalized based on the amount of RNA input into the RT reaction.
4
Quantitative gene expression analysis
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PolyA mRNA was isolated using QuickPick mRNA magnetic beads (Bio-Nobile, Finland). Four replicate RNA pools from each substrate type were reverse transcribed with random hexamer primers using GeneAmp Gold RNA PCR Reagent Kit (Applied Biosystems). The resultant first-strand cDNA was analyzed in duplicate PCR reactions using iQ Supermix kit (Bio-Rad Laboratories) and FAM-labeled TaqMan Gene Expression Assays (Applied Biosystems) for bone sialoprotein (BSP; Rn00561414_m1), osteocalcin (OC; Rn00566386_g1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a control gene; Rn99999916_s1). PCRs were carried out using an iCycler iQ real-time PCR detection system with software version 3.1 (Bio-Rad Laboratories). The following cycling conditions were used: 95°C/5 min; 40 cycles of 95°C/20 s, 60°C/60 s. Target gene expression was first normalized to the expression of the housekeeping gene GAPDH in the same sample (ΔCt) and then converted to a fold ratio as compared to the average baseline expression of that target gene measured in P0 group at 7 days (ΔΔCt). Finally, the 2−ΔΔCt method was used to convert normalized gene expression levels to fold differences and statistics were calculated on these values [15 (link)].
5
Quantification of RNA Transcripts
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Total RNA was extracted with TRIzol Reagent (Ambion, CA). The concentration and purity of the extracted RNA was measured by a NanoDrop Spectrophotometer (Thermo, MA) and an Agilent 2100 Bioanalyzer (Agilent, CA). To quantify specific miRNA and circRNA content, a TaqMan MiRNA assay kit (Applied Biosystems, CA), an iScript Select cDNA Synthesis kit (Bio-Rad, CA), and an iQSupermix kit (Bio-Rad, CA) were used to enable target-specific detection, and the procedures complied with the manufacturer’s instructions. To quantify mRNA levels, an iScript Select cDNA Synthesis kit was also adopted to produce complimentary DNA transcripts with oligo-dT primers. Based on the primer set specialized for target mRNA, iQSYBR Green mix (Bio-Rad) was applied to enable fragment amplification by real-time quantitative reverse transcription PCR (RT-qPCR). The reactions were carried out on a PCR flatform (Applied Biosystems) that also enabled capturing of measurements. The raw quantitative results were normalized to the level of β-Actin, which was followed by data processing with the comparative Ct (ΔΔCt) method (2 −ΔΔCt with logarithm transformation) to estimate the relative expression levels [45 (link)].
6
Quantifying HIV-1 Proviral Integration
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Integration of viral DNA into the genomic DNA of HOKs was determined using an Alu-long terminal repeat (LTR)-based real-time nested-PCR assay47 (link). Two human genomic Alu forward primers (Alu1 and Alu2) and an HIV-1 LTR reverse primer extended with a lambda phage-specific heel sequence at the 5′ end of the oligonucleotide (M667–L) were used to enrich host genomic DNA with downstream proviral DNA using PCR. The integrated HIV-1 provirus was then quantified using an iQ Supermix kit (Bio-Rad) with a reaction solution containing a lambda-specific primer (Lambda T) and an ltr primer (AA55M) (Table S1 ), and the PCR products from the first-round of PCR. Amplification of human gapdh gene was used for normalizing levels of HIV-1 provirus.
7
Quantification of miRNA Levels by qRT-PCR
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Through treatment of the TRIzol reagent, RNA was extracted from the serum or cultured cells. According to the protocol, the quantitative determination of miRNAs was conducted using the TaqMan miRNA assay kit (Applied Biosystems, Inc., Carlsbad, CA, USA), iScript Select cDNA synthesis kit (Bio-Rad, Hercules, CA, USA), and iQSupermix kit (Bio-Rad). The complementary DNA (cDNA) was synthesized using oligo-dT primers and iScript Select cDNA synthesis kit (Bio-Rad). Real-time PCR was analyzed by applying certain primer profile and iQSYBR Green mix (Bio-Rad). U6 (Yu et al. 2015) and GAPDH were used to standardize miRNA and gene-specific expression levels, respectively. The relative gene expression was measured based on the 2 -ΔΔCt method (Livak and Schmittgen 2001) . Primers used for PCR are listed in Table 1.
Western blot assay. The separation of protein from AC16 cells were achieved using radioimmunoprecipitation assay reagent (Abcam, Cambridge, MA, USA). Protein concentration was quantified by the bicinchoninic acid kit (Beyotime). Then, 30 μg protein was loaded in sodium dodecyl sulfate polyacrylamide gel for separation and then transferred onto polyvinylidene fluoride membranes. After a blockade with 5% non-fat milk, membrane incuba-
Western blot assay. The separation of protein from AC16 cells were achieved using radioimmunoprecipitation assay reagent (Abcam, Cambridge, MA, USA). Protein concentration was quantified by the bicinchoninic acid kit (Beyotime). Then, 30 μg protein was loaded in sodium dodecyl sulfate polyacrylamide gel for separation and then transferred onto polyvinylidene fluoride membranes. After a blockade with 5% non-fat milk, membrane incuba-
8
Quantitative Analysis of RNA Molecules
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Total RNA was extracted with TRIzol Reagent (Ambion, CA). The concentration and purity of the extracted RNA was measured by a NanoDrop Spectrophotometer (Thermo, MA) and an Agilent 2100 Bioanalyzer (Agilent, CA). To quantify specific miRNA and circRNA content, a TaqMan MiRNA assay kit (Applied Biosystems, CA), an iScript Select cDNA Synthesis kit (Bio-Rad, CA), and an iQSupermix kit (Bio-Rad, CA) were used to enable target-specific detection, and the procedures complied with the manufacturer's instructions. To quantify mRNA levels, an iScript Select cDNA Synthesis kit was also adopted to produce complimentary DNA transcripts with oligo-dT primers. Based on the primer set specialized for target mRNA, iQSYBR Green mix (Bio-Rad) was applied to enable fragment amplification by real-time quantitative reverse transcription PCR (RT-qPCR). The reactions were carried out on a PCR flatform (Applied Biosystems) that also enabled capturing of measurements. The raw quantitative results
were normalized to the level of β-Actin, which was followed by data processing with the comparative Ct (ΔΔCt) method (2 -ΔΔCt with logarithm transformation) to estimate the relative expression levels. 28
were normalized to the level of β-Actin, which was followed by data processing with the comparative Ct (ΔΔCt) method (2 -ΔΔCt with logarithm transformation) to estimate the relative expression levels. 28
9
Quantitative Real-Time PCR for mRNA and miRNA
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