RNA extraction and quantitative RT-PCR were performed as previously described [27 (link)] from mice fasted overnight using the E.Z.N.A. Total RNA Kit II (Omega Bio-tek Inc., Norcross, GA). Primer sequences (Integrated DNA Technologies, Coralville, IA) can be found in Supplemental Table 1 . Gene expression levels were evaluated by the ΔΔ-Ct method [35 (link)] with GAPDH used to control for total mRNA recovery; control values were normalized to a group mean of 1.0.
Primer sequences
Manufactured by Integrated DNA Technologies
Sourced in United States
Primer sequences are short, synthetic DNA molecules designed to serve as starting points for DNA amplification and sequencing. They are essential components of various molecular biology techniques, such as polymerase chain reaction (PCR) and DNA sequencing. Primer sequences provide the necessary information for DNA polymerase to initiate the replication process, enabling the targeted amplification or sequencing of specific DNA regions.
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Lab products found in correlation
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Total rna kit 2, by Omega Bio-Tek (1 mentions)
Lightcycler 480 instrument, by Roche (1 mentions)
Sybr green 1, by Roche (1 mentions)
7900ht real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Iscript cdna kit, by Bio-Rad (1 mentions)
Singleshot cell lysis kit, by Bio-Rad (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
6 protocols using primer sequences
1
Quantitative RT-PCR of Fasted Mice
2
Quantitative Analysis of Chromatin Immunoprecipitation
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Based on our previous findings [13 ,14 (link),15 (link)], in quantitative PCR (qPCR) we amplified satellite II (Sat II) elements. Primer sequences (Integrated DNA technologies, Coralville, IA, USA) used for Sat II were 5′-CATCGAATGGAAATGAAAGGAGTC-3’ (F) and 5′-ACCATTGGATGATTGCAGTCAA-3’ (R) [25 (link)].
Three-microliter (out of 30 µL, precipitated from 200 µL plasma) ChIP-DNA were subjected to qPCR, which was performed in the LightCycler 480 Instrument (Roche Diagnostics) using SYBR Green I (Roche Diagnostics) as the fluorescence molecule. We used a gradual PCR program with annealing temperature starting at 60 °C for 2 cycles followed by 38 cycles at 55 °C. Samples with a threshold cycle (Ct) >40 were considered negative. Amplification of the appropriate product was confirmed by melting curve analysis following amplification. To estimate the amounts of H3K9me3- and H4k20me3-related Sat II sequences in immune precipitated fragments, we subtracted non-Ig values from qPCR values of each sample and performed an absolute quantification using a dilution series of a sample with known DNA concentration.
Three-microliter (out of 30 µL, precipitated from 200 µL plasma) ChIP-DNA were subjected to qPCR, which was performed in the LightCycler 480 Instrument (Roche Diagnostics) using SYBR Green I (Roche Diagnostics) as the fluorescence molecule. We used a gradual PCR program with annealing temperature starting at 60 °C for 2 cycles followed by 38 cycles at 55 °C. Samples with a threshold cycle (Ct) >40 were considered negative. Amplification of the appropriate product was confirmed by melting curve analysis following amplification. To estimate the amounts of H3K9me3- and H4k20me3-related Sat II sequences in immune precipitated fragments, we subtracted non-Ig values from qPCR values of each sample and performed an absolute quantification using a dilution series of a sample with known DNA concentration.
3
Quantitative RNA Expression Analysis
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Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA) following the manufacturer's instructions and quantitative RT‐PCR used the iTaq Universal SYBR Green Supermix (Bio‐Rad, Berkeley, California) and a 7900HT real‐time PCR instrument (Applied Biosystems, Foster City, CA) as previously described11 (see Supporting Information). Primer sequences (Integrated DNA Technologies, USA) are in Table S4 , Supporting Information.
4
Real-time PCR Gene Expression Analysis
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The relative mRNA expression was measured in triplicate via the comparative cycle threshold method using a 7500 real-time PCR system (Applied Biosystems). The primer sequences (Integrated DNA Technologies) are shown in Supplementary Table 1 . We used the housekeeping gene β-actin as the internal standard.
5
qRT-PCR Analysis of BMSC and AMSC
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To quantify relative mRNA transcript copy numbers of selected genes, 24 h after BMSC and AMSC transfection with the LF 3000 as described earlier, cell lysate was prepared using the SingleShot Cell Lysis Kit (Bio-Rad, Hercules, CA, USA), and RNA was reverse transcribed using the iScript cDNA Kit (Bio-Rad). qRT-PCR was performed on a QuantStudio 6 Flex Real-Time PCR System (Thermo) with Power SYBR Green Master Mix (Thermo Fisher Scientific), and expression was calculated by the ΔΔCt method normalizing to endogenous controls RPL13A and HPRT1. See Table S2 for primer sequences (Integrated DNA Technologies).
6
ChIP Assay for TCF7L2 Binding
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ChIP assays were performed using a SimpleChIP Enzymatic Chromatin IP kit (#9003; Cell Signaling Technologies). Briefly, cells were crosslinked using 37% formaldehyde at a final concentration of 1% for 10 min at room temperature and then quenched with 125 mM glycine for 5 min. Cells were washed twice with cold PBS, harvested in PBS with protease inhibitor cocktail (PIC) and lysed to get nuclei. Chromatin was harvested, fragmented using micrococcal nuclease to length of approximately150-900 bp and then briefly sonicated to lyse nuclear membranes. The digested chromatin (500 μg) was subjected to immunoprecipitation with specific TCF7L2 antibody (Celling Signaling) or anti-Rabbit IgG (#2729 Cell signaling) as negative control overnight at 4° C with rotation and followed by ChIP-Grade protein G magnetic beads incubated for 2 hours at 4° C with rotation. After immunoprecipitation, the chromatin was eluted from the antibody/protein G magnetic beads and cross-links were reversed. DNA was purified and analyzed by RT-qPCR.
Primer sequences (Integrated DNA Technologies) for the Bmp4 binding site were as follows:
forward primer 5'-GGTACCTGCACTTAAGCTTTGTCGG 3′, and Reverse primer: 5'-TCGTAGTCGCTGCACGCAG-3′. The data were quantified by percent input and performed in triplicates.
Primer sequences (Integrated DNA Technologies) for the Bmp4 binding site were as follows:
forward primer 5'-GGTACCTGCACTTAAGCTTTGTCGG 3′, and Reverse primer: 5'-TCGTAGTCGCTGCACGCAG-3′. The data were quantified by percent input and performed in triplicates.
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