The quantification of the mRNA levels of the housekeeping genes GAPDH and β-ACTIN, pluripotency genes OCT4 and NANOG, GC markers FRAGILIS, PIWIL2 and STELLA, SSC markers UCHL1 and CD90 and male GC markers DAZL and STRA8, was determined using Q-PCR (Table 1 ). Total RNA was isolated from cells using a Quick-RNA MiniPrep kit (Zymo Research) following the manufacturer’s instructions. Total RNA was quantified using a Qubit 3.0 (Invitrogen, Fluorometer, CA, USA). Genomic DNA digestion was performed using DNase I from the Quick-RNA MiniPrep kit (Zymo Research) following the manufacturer’s instructions. The cDNA was synthesized and amplified using an Affinity Script Q-PCR cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA, USA), using a Step One thermocycler (Applied Biosystems, Foster City, CA, USA). The PCR reaction was performed using a Brilliant SYBR Green QPCR Master Mix kit (Agilent Technologies) and an Eco Real-Time PCR System thermocycler (Illumina, San Diego, CA, USA). Each reaction tube consists of 5 μL Sybr Green, 1 μL forward primer, 1 μL reverse primer, 2 μL nuclease-free H2O and 5 ng cDNA. The cDNA amplification was extended for 40 cycles, and relative expression analysis was performed using the ΔΔCt (Ct: threshold value) method normalized with both GAPDH and β-ACTIN housekeeping genes [30 (link)].
Brilliant sybr green kit qpcr master mix
Manufactured by Agilent Technologies
Sourced in United States
The Brilliant SYBR Green QPCR Master Mix is a ready-to-use solution for performing quantitative real-time PCR (qPCR) experiments. It contains SYBR Green I dye, which binds to double-stranded DNA, allowing for the detection and quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Mulv reverse transcriptase pcr kit, by Thermo Fisher Scientific (3 mentions)
Quick rna miniprep kit, by Zymo Research (2 mentions)
Qubit 3, by Thermo Fisher Scientific (2 mentions)
Qiashredder colums, by Qiagen (2 mentions)
Mx3005p, by Agilent Technologies (2 mentions)
Stepone thermocycler, by Thermo Fisher Scientific (1 mentions)
Affinityscript qpcr cdna synthesis kit, by Agilent Technologies (1 mentions)
Quick rna miniprep, by Zymo Research (1 mentions)
Eco real time pcr system, by Illumina (1 mentions)
Stepone thermal cycler, by Thermo Fisher Scientific (1 mentions)
Stratagene mx3005p, by Agilent Technologies (1 mentions)
Qiashredder column, by Qiagen (1 mentions)
5 protocols using brilliant sybr green kit qpcr master mix
1
Quantification of Stem Cell Genes
2
Quantification of mRNA Expression by Q-PCR
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Quantification of mRNA levels was determined by Q-PCR (Table 1 ). Total RNA was isolated from the cells using a Quick-RNA MiniPrep (Zymo Research) kit following manufacturer’s instructions. Total RNA was quantified using a Qubit 3.0 (Invitrogen, Fluorometer, CA, USA). DNA genomic digestion was performed using DNAsa I from the Quick-RNA MiniPrep kit (Zymo Research) following the manufacturer’s instructions. The cDNA was synthesized and amplified using a cDNA synthesis kit Q-PCR from Affinity Script (Agilent Technologies, Santa Clara, CA, USA), using a Step One thermal cycler (Applied Biosystems, Foster City, CA, USA). The PCR reaction was performed using a Brilliant SYBR Green QPCR Master Mix kit (Agilent Technologies) and an Eco Real-Time PCR System (Illumina, San Diego, CA, USA). Each reaction tube consists of 5 μL Sybr Green, 1 μL Foward primer, 1 μL Reverse primer, 2 μL nuclease-free H2O, and 5 ng cDNA. The amplification of cDNA was extended over 40 cycles, and relative expression analysis was performed using the ΔΔCt (Ct: Threshold Value) method normalized with GAPDH and β-ACTIN gene expression [32 (link)].
3
Chondrocyte RNA Extraction and Analysis
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RNA extraction was performed from mouse primary chondrocytes using RNeasy Mini Kit (Qiagen, Invitrogen) and QIAshredder colums (Qiagen, Invitrogen), following the manufacturer’s protocol. RNA reverse transcription was performed using the MuLV Reverse Transcriptase PCR Kit (Applied Biosystems). After RNA reverse transcription to cDNA, real time PCR reactions were performed for a relative quantification of collagen X, MMP-13 and IL-6 performed on a Stratagene Mx3005P (Agilent Technologies, CA, USA) with Brilliant SYBR Green Kit QPCR Master Mix (Stratagene, Agilent Technologies, CA, USA), according to the manufacturer’s protocol.
4
Chondrocyte Gene Expression Analysis
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RNA extraction was performed from mouse primary chondrocytes using RNeasy Mini Kit (Qiagen, Invitrogen) and QIAshredder colums (Qiagen, Invitrogen), following the manufacturer's protocol. RNA reverse transcription was performed using the MuLV Reverse Transcriptase PCR Kit (Applied Biosystems). After RNA reverse transcription to cDNA, real time PCR reactions were performed for a relative quantification of COL10a1 (forward: 5′-TTCTGCTGCTAATGTTCTTGACC-3′; reverse: 5′-GGGATGAAGTATTGTGTCTTGGG-3′), Mmp-13 (forward: 5′-TGTTTGCAGAGCACTACTTGAA-3′; reverse: 5′-CAGTCACCTCTAAGCCAAAGAAA-3′), ENTPD1 (forward:5′-ACAAGGGCTGCGAGATAAGA-3′; reverse: 5′-CCACCCAGACCTGTTGACTT-3′), NT5E (forward: CAAATCCCACACAACCACTG-3′; reverse: 5′-TGCTCACTTGGTCACAGGAC-3′), PANX1 (forward: 5′-CCACCGAGCCCAAGTTCAA-3′; reverse: 5′-CCGGGTTGTTGAGTGTTACAG-3′), SLC29A1 (forward: 5′-CCAGTGGTTCTGAGCTGTCA-3′; reverse: 5′-CTGTTGGTGGGTGGAGAGTT-3′), SLC29A2 (forward: 5′-GCTGGGTACCATGCCTTCTA-3′; reverse: 5′-CCACACAGGGTGTGATGAAG-3′) and ANKH (forward: 5′-CAAGAGAGACAGGGCCAAAG-3′; reverse: 5′-AAGGCAGCGAGATACAGGAA-3′) performed on a Stratagene Mx3005P (Agilent Technologies) with Brilliant SYBR Green Kit QPCR Master Mix (Stratagene, Agilent Technologies), according to the manufacturer's protocol.
5
Quantification of Autophagy-related Genes
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Total RNA from was extracted from TC28α2 cells using an RNeasy Mini Kit (Qiagen, Invitrogen) and QIAshredder columns (Qiagen, Invitrogen), following the manufacturer’s protocol. RNA reverse transcription was carried out using the MulV Reverse Transcriptase PCR Kit (Applied Biosystems). Upon conversion to cDNA, real time PCR reactions were performed for relative quantification of P62/SQSTM1(forward: 5′-TGCCCAGACTACGACTTGTG-3′; reverse: 5′-AGTGTCCGTGTTTCACCTTCC-3′), BECN1 (forward: 5′-ACAGTGGACAGTTTGGCACA-3′; reverse: 5′-CGGCAGCTCCTTAGATTTGT-3′), and GABARAPL1 (forward: 5′-AGGAGGACCATCCCTTTGAGT-3′; reverse: 5′-TGGCCAACAGTAAGGTCAGA-3′) as compared to control GAPDH (forward: 5′-GACATCAAGAAGGTGGTGAA-3′; reverse: 5′-TGTCATACCAGGAAATGAGC-3′). RT-PCR was performed on a Stratagene Mx3005P (Agilent Technologies) with Brilliant SYBR Green Kit QPCR Master Mix (Stratagene, Agilent Technologies), according to the manufacturer’s protocol.
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