Total RNA was extracted from CEP chondrocytes using the total RNA extraction kit (Toyobo, Japan), following the manufacturer’s instructions. RNA purity and concentration were measured using a microvolume spectrophotometer (Thermo Fisher Scientific, Logan, UT, United States); 1 μg of total RNA was reverse transcribed into cDNA using First Strand cDNA Synthesis Kit (Toyobo, Osaka, Japan). Real-time PCR was performed under following cycling conditions: 30s of polymerase activation at 95°C, followed by 40 cycles of 95°C for 5s and 60°C for 30s. Sequences of primers for the reference gene (GAPDH) and interested genes are listed as follows: TNFα (F), 5′ -GACCCCTTTACTCTGACCCC- 3′, (R) 5′ -AGGCTCCAGTGAATTCGGAA-3′; IL-1β (F), 5′ -ACA- GATGAAGTGCTCCTTCCA-3′, (R) 5′ -GTCGGAGATTCGTAGCTGGA-3′; IL-6 (F), 5′ -TGTCTTCCTCACCGATTCCT-3′, (R) 5′ -ACCACCC- GAGCTCTGTCTTACTC-3′; and GAPDH (F) 5′ -AGGTCGGTGTGAACGGATTTG-3′, (R) 5′ -TGTAGACCATGTAGTTGAGGTCA-3′;
Microvolume spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Microvolume Spectrophotometer is a compact, high-precision instrument that measures the absorbance of small sample volumes, typically ranging from 0.5 to 2 microliters. It is designed to quantify and analyze the concentration of nucleic acids, proteins, and other biomolecules in a wide range of applications, including molecular biology, biochemistry, and life science research.
Automatically generated - may contain errors
Lab products found in correlation
Total rna extraction kit, by Toyobo (2 mentions)
First strand cdna synthesis kit, by Toyobo (1 mentions)
Strand cdna synthesis kit, by Toyobo (1 mentions)
Sybr green realtime pcr master mix, by Toyobo (1 mentions)
Superscript 3 one step kit, by Thermo Fisher Scientific (1 mentions)
Chelex 100, by Bio-Rad (1 mentions)
Mutanolysin, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
5 protocols using microvolume spectrophotometer
1
Quantifying Inflammatory Cytokine Expression
2
Quantifying Iron-related Gene Expression
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RT-PCR was used to quantify gene expression. Total RNA (mRNA) was extracted from articular chondrocytes using the total RNA extraction kit (Toyobo, Japan) following the manufacturer's instructions. RNA concentration and purity were assessed using a Microvolume Spectrophotometer (Thermo Fisher Scientific, Logan, UT, USA). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using first Strand cDNA Synthesis Kit (Toyobo, Osaka, Japan). Then the cDNA was amplified by SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) with following cycling conditions: 30 s of polymerase activation at 95°C, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. GAPDH was used as an internal control. Each cDNA sample was run in triplicate. Sequences of primers for the reference gene (GAPDH) and interested genes are listed as follows: DMT1, Forward: 5′-TCTTCGGTTCCTCTCACTCCTGTG-3′, Reverse:5′-AGAGCCTGCCACCACCAGTC-3′; TFR1, Forward:5′-AATGGTTCGTACAGCAGCGGAAG-3′, Reverse:5′-TAGCACGGAAGTAGTCTCCACGAG-3′; FPN, Forward:5′-GCCTCTGCCTCTGCCTCTACC-3′, Reverse: 5′-AGTCAGTCCACGAGTGCTACAGTC-3′; GAPDH:Forward:5′-CTCCCACTCTTCCACCTTCG-3′, Reverse:5′-TTGCTGTAGCCGTATTCATT-3′
3
Quantifying Metanephric Gene Expression
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mRNA from metanephroi was isolated using RNA extraction kit (Isogen, Nippon Genes), followed by RNA quantification with microvolume spectrophotometer (Thermo). An appropriate amount of mRNA was reverse transcribed to cDNA and amplified with SuperScript III One-Step kit (Invitrogen) by using primers of Flk-1 (sense: 5′-GCCCTGCTGTGGTCTCACTAC and antisense: 5′-CAAAGCATTGCCCATTCGAT) [22] (link), c-kit (sense: 5′-TTATCCTTTAGGCCGTGTGG and antisense: 5′-TGTGGCCCTTAAGTACCTG) [23] , Flt-1 (sense: 5′-GAGGAGGATGAGGGTGTCTATAGGT and antisense: 5′-GTGATCAGCTCCAGGTTTGACTT), Tie-2 (sense: 5′-ATGTGGAAGTCGAGAGGCGAT and antisense: 5′-CGAATAGCCATCCACTATTGTCC) [22] (link), PECAM-1 (sense: 5′-GAGCCCAATCACGTTTCAGTTT and antisense: 5′-TCCTTCCTGCTTCTTGCTAGCT) [22] (link), and the primers of GAPDH (sense: 5′-TGTTCCTACCCCCAATGTGT and antisense: 5′-TGTGAGGGAGATGCTCAGTG) [24] (link). GAPDH was used as internal control. The cDNA amplification was carried out at 94 °C for 30sec, 52 °C for 30 s, 68 °C for 1 min, followed by cooling at 4 °C.
4
DNA Extraction from Embryo Brain
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10–20 mg of day 9 post-hatching embryo brain tissue was pipetted and lysed in 150 μl of lysis buffer containing 10% of chelating beads (Chelex 100 Biorad), 0.2% SDS, 10mM Tris-HCl pH 8, 0.2 μg/μl Proteinase K. Brain was chosen for DNA extraction since it is a soft tissue easy to aspirate with a P1000 pipette. Tissues were incubated for 3h at 55°C following by 15 minute incubation at 95°C. Samples were then centrifuged for 5 minutes at 13000g at room temperature. Supernatant were recovered and stored at -20°C until used. For sensitivity assessment, DNA lysate quantification was performed with Qubit Fluorometer (dsDNA HS (high sensitivity) Assay Kit, Invitrogen) and by reading the 260 nm absorbance with a micro volume spectrophotometer (Nanodrop One Thermo Scientific, Wilmington USA) to estimate the purity of the sample.
5
Genomic DNA Extraction from Streptococcus pyogenes
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Genomic DNA was extracted from the Streptococcus pyogenes isolates using a silica-membrane spin column kit (Macherey-Nagel). In brief, overnight cultures (3.5 mL) were harvested by centrifugation at 3500 x g, resuspended in ice-cold 70% ethanol and incubated at -20 °C for 20 minutes. The cell wall was digested by resuspending the bacteria in 25 mM Tris-HCl, 2 mM EDTA, 1% (v/v) Triton X-100 containing 20 mg/mL lysozyme and 250 units/mL mutanolysin (both enzymes from Sigma-Aldrich) followed by incubation at 37 °C for 2 hours.
Genomic DNA was released from the bacteria by resuspending the bacteria in a buffer containing SDS and 20 mg/mL proteinase K and overnight incubation at 56 °C. Subsequent DNA purification was performed according to the manufacturer's protocol for the silica-membrane spin column kit. Preheated elution buffer (70 °C, 5 mM Tris-HCl, pH 8.5) was applied to the spin column followed by incubation of the spin column at 70 °C for 10 minutes prior to elution of the DNA. The quantity and quality of the extracted genomic DNA were assessed using agarose gel electrophoresis, a microvolume spectrophotometer (Thermo Scientific) and a fluorescence-based quantification kit (Life Technologies). The purified genomic DNA was sent to GATC (Germany) for genomic library construction and sequencing on a HiSeq 2000 (Illumina) with 50 bp single reads.
Genomic DNA was released from the bacteria by resuspending the bacteria in a buffer containing SDS and 20 mg/mL proteinase K and overnight incubation at 56 °C. Subsequent DNA purification was performed according to the manufacturer's protocol for the silica-membrane spin column kit. Preheated elution buffer (70 °C, 5 mM Tris-HCl, pH 8.5) was applied to the spin column followed by incubation of the spin column at 70 °C for 10 minutes prior to elution of the DNA. The quantity and quality of the extracted genomic DNA were assessed using agarose gel electrophoresis, a microvolume spectrophotometer (Thermo Scientific) and a fluorescence-based quantification kit (Life Technologies). The purified genomic DNA was sent to GATC (Germany) for genomic library construction and sequencing on a HiSeq 2000 (Illumina) with 50 bp single reads.
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