The ESR1 gene polymorphisms rs9340799, rs2234693, and rs3798759, and ESR2 gene polymorphisms rs207764, rs4986938, and rs1256049 were selected, as described in previous studies [18 (link),19 (link),22 (link),23 (link),25 (link)]. Peripheral blood samples were obtained from all subjects by using the QlAamp DNA Mini Kit (Qiagen, Hilden, Germany). Multiplex polymerase chain reaction (PCR) was performed on a GeneAmp 9700 PCR thermocycler (Applied Biosystems, Foster City, California, USA). Genotyping of the ESR1 and ESR2 polymorphisms was determined by direct sequencing. The 3730XL genetic analyzer (Applied Biosystems, Foster City, California, USA) was used for sequencing. The GeneScan™ version 3.7 Software (Applied Biosystems, Foster City, California, USA) was used for data analysis.
Geneamp pcr 9700 thermocycler
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneAmp PCR 9700 Thermocycler is a laboratory instrument designed for conducting polymerase chain reaction (PCR) experiments. It precisely controls the temperature and cycling parameters required for DNA amplification.
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Lab products found in correlation
3730xl genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Qlaamp dna mini kit, by Qiagen (1 mentions)
Genescan 3, by Thermo Fisher Scientific (1 mentions)
Hotstar taq polymerase, by Qiagen (1 mentions)
Exosap it, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht, by Thermo Fisher Scientific (1 mentions)
Hotstar hifidelity polymerase kit, by Qiagen (1 mentions)
Abi 3730 dna analyzer, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Taqman mirna assay system, by Thermo Fisher Scientific (1 mentions)
Sybr green real time pcr master mix, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Non acetylated bovine serum albumin, by Merck Group (1 mentions)
Geneamp pcr buffer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
15 protocols using geneamp pcr 9700 thermocycler
1
Genotyping of ESR1 and ESR2 polymorphisms
2
Genotyping of MC2R Polymorphisms
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SNP selection was performed using Haploview software (https://sourceforge.net/projects/haploview/ ) choosing only SNPs
with a minor allele frequency higher than 0.05 and r2≥0.8 based on
the HapMap database (CHB, Chinese Han population) (http://coriell.org/1/NHGRI/Collections/HapMap-Collections/HapMap-Project ).
After screening, seven tag SNPs (rs16941303, rs16941314, rs2186944, rs28926188,
rs7230126, rs948322, and rs948331) in the MC2R gene were selected. Genomic DNA
was extracted from peripheral blood leukocytes using the standard
phenol-chloroform method. Multiplex polymerase chain reaction (PCR) assays were
performed on a GeneAmp 9700 PCR thermocycler (Applied Biosystems, Foster City,
California, USA). The reactions were performed in a total volume of 10 µL,
including 1 µL of genomic DNA, 1 µL of primer mix (1 µM of each primer), and 1 U
of Hotstar Taq polymerase (Qiagen Inc., Valencia, CA, USA). The cycling
parameters were as follows: 95°C for 2 minutes; 10 cycles at 95°C for 20 s, 65°C
for 40 s, and 72° for 30 s; 25 cycles at 95°C for 20 s, 55°C for 30 s, and 72°C
for 1 minute; then the samples were held at 4°C. The genotypes of the MC2R
polymorphisms were determined by Sanger sequencing. A 3730XL genetic analyzer
(Applied Biosystems) was used for sequencing. GeneScan ™3.7 software (Applied
Biosystems) was used for data analysis.
with a minor allele frequency higher than 0.05 and r2≥0.8 based on
the HapMap database (CHB, Chinese Han population) (
After screening, seven tag SNPs (rs16941303, rs16941314, rs2186944, rs28926188,
rs7230126, rs948322, and rs948331) in the MC2R gene were selected. Genomic DNA
was extracted from peripheral blood leukocytes using the standard
phenol-chloroform method. Multiplex polymerase chain reaction (PCR) assays were
performed on a GeneAmp 9700 PCR thermocycler (Applied Biosystems, Foster City,
California, USA). The reactions were performed in a total volume of 10 µL,
including 1 µL of genomic DNA, 1 µL of primer mix (1 µM of each primer), and 1 U
of Hotstar Taq polymerase (Qiagen Inc., Valencia, CA, USA). The cycling
parameters were as follows: 95°C for 2 minutes; 10 cycles at 95°C for 20 s, 65°C
for 40 s, and 72° for 30 s; 25 cycles at 95°C for 20 s, 55°C for 30 s, and 72°C
for 1 minute; then the samples were held at 4°C. The genotypes of the MC2R
polymorphisms were determined by Sanger sequencing. A 3730XL genetic analyzer
(Applied Biosystems) was used for sequencing. GeneScan ™3.7 software (Applied
Biosystems) was used for data analysis.
3
DNA Sequencing Protocol with ExoSAP-IT Cleanup
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In order to perform DNA sequencing, the amplified PCR products were enzymatically cleaned before cycle sequencing, 3 μL of ExoSAP-IT (USB Corporation, Cleveland, OH, USA) was added to 5 μL of each amplified PCR product, as described above [13 (link)]. The mixture was incubated at 37 °C for 20 min followed by 80 °C for 15 min on a GeneAmp PCR 9700 Thermocycler (Applied Biosystems, Foster City, CA, USA). The purified PCR products were sequenced using AB Big-Dye 3.1 dye chemistry and AB 3500 XL automated DNA sequencers (Applied Biosystems) with sequencing reaction competed for 25 cycles (each cycle is 96 °C for 30 s, 50 °C for 15 s, and 60 °C for 4 min) and held at 4 °C in a GeneAmp PCR 9700 Thermocycler (Applied Biosystems). The cycle sequencing reactions contained 2 μL of cleaned PCR product, 1 μL of BigDye Terminator v3.1 Ready Reaction Mix, 2 μL of 5× Sequencing Buffer, 1.6 pmol of Forward or Reverse sequencing primer, and water in a final volume of 20 μL. Sequencing reactions were cleaned up with the Performa® DTR Gel Filtration Cartridges following manufacturer’s protocol (Edge Bio, Gaithersburg, MD, USA). Sequence accuracy was confirmed by performing two-directional sequencing. Multiple alignments of the generated nucleotide sequences were carried out by using the BioEdit and Geneious programs with manual adjustments.
4
Quantifying Murine miR-142a and miR-21 Expression
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All reagents, primers, and probes were obtained from Applied Biosystems. A U6 endogenous control was used for normalization. Reverse transcriptase reactions and real-time PCR were performed according to the manufacturer’s protocols (Applied Biosystems). RNA concentrations were determined with a NanoDrop instrument (NanoDrop Technologies). One nanogram of RNA per sample was used for the assays. All RT reactions, including no-template controls and RT minus controls, were run in triplicate in GeneAmp PCR 9700 Thermocycler (Applied Biosystems). Gene expression levels were quantified using the ABI Prism 7900HT sequence detection system (Applied Biosystems). Relative expression was calculated using the comparative threshold cycle (Ct) method. The primers used for target genes: murine miR-142a (fwd):5′-TGGCATGAGGATCAGCAGGG-3′, murine miR-142a (rev):5′–GGCAGTCCGCAGCTCTAGG-3′; murine miR-21 (fwd): 5′-GCGTGCTAATGGTGGA-3′, murine miR-21 (rev): 5′-CAGGCGTATCAGTGGG-3′.
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was subjected to quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR). mRNAs were analyzed using TaqMan Gene Expression Assays in accordance with the manufacturer’s instructions (Applied Biosystems, Foster City, CA). All RT reactions were run in a GeneAmp PCR 9700 Thermocycler (Applied Biosystems). Probes used for the analyses were as follows: KDM5B, Hs00981910_m1; MYC, Hs00905030_m1 (Applied Biosystems), HBV proteins, shown in Table 1 . The experiments were performed in triplicate. The TaqMan gene assay for actin was used to normalize the relative abundance of mRNA.
6
Whole Genome Sequencing of Adenovirus
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The hexon and fiber genes of HAdV from tissue culture were amplified as described previously.12 As the use of whole genome sequencing is advised to confirm the type of HAdV,2 (link), 13 (link) the whole genome of one isolate in this study was sequenced using 41 sets of primers designed in-house. In brief, 41 pairs of M13F/M13R-tailed primers for the whole genome were used to perform separate PCRs. The product of each primer had overlapping sequences of 200–300 bp. PCR amplification of the hexon and fiber genes and whole genome sequencing segments was performed with HotStar HiFidelity Polymerase Kit (Qiagen, Germany); 0.8 μM of each primer was used. PCRs were performed in a GeneAmp PCR-9700 thermocycler (Applied Biosystems, USA) as follows: for the hexon gene, 95 °C for 15 min, followed by 30 cycles of 45 s at 94 °C, 45 s at 60 °C, 90 s at 72 °C, and a final extension step of 72 °C for 10 min; for the fiber gene and whole genome sequencing fragments, 95 °C for 15 min, followed by 30 cycles of 45 s at 94 °C, 45 s at 46 °C, 60 s at 72 °C, and a final extension step of 72 °C for 10 min.
The PCR products were analyzed with a QIAxcel Nucleic Acid Analyzer (Qiagen, Germany), purified using the QIAquick PCR Purification Kit (Qiagen, Germany), and then sequenced by Invitrogen Biotechnology Co. Ltd in Shanghai, China using an ABI 3730 DNA Analyzer (Applied Biosystems, USA).
The PCR products were analyzed with a QIAxcel Nucleic Acid Analyzer (Qiagen, Germany), purified using the QIAquick PCR Purification Kit (Qiagen, Germany), and then sequenced by Invitrogen Biotechnology Co. Ltd in Shanghai, China using an ABI 3730 DNA Analyzer (Applied Biosystems, USA).
7
Quantification of miRNA Expression
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Total RNA was extracted from cultured cells, using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For the preparation of cellular miRNAs, small-sized RNAs containing miRNAs were isolated from total RNA, using the RNeasy MinElute Cleanup kit (Qiagen, Valencia, CA, USA). To evaluate miRNA expression levels, we used single-tube TaqMan miRNA assays as previously described (Applied Biosytems, Franklin Lakes, NJ, USA) (29 (link), 30 (link)). Normalization was performed with 18S. All reverse transcription reactions, no-template controls, and RT primer minus controls were run in a GeneAmp PCR 9700 Thermocycler (Applied Biosystems). Gene expression levels were quantified using the ABI 7300 Real-Time PCR System (Applied Biosytems). Comparative real-time PCR was performed in triplicate, including the no-template controls, using specific primers for miR-9, with all reagents obtained from Applied Biosytems. The TaqMan RNU6B endogenous control kit was purchased from Applied Biosystems. PCR was carried out at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative expression was calculated using the comparative threshold (Ct) method.
8
Quantification of miR-506 and STAT3 mRNA
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Total RNA, including miRNA, was isolated from the isolated primary NK cells or NK-92 cells using TRIzol reagent (Invitrogen). Complementary DNA was synthesized from 1 µg of total RNA using PrimeScript RT reagent kit (TaKaRa, Dalian, China). miR-506 detection was performed using TaqMan miRNA assay system (Applied Biosystems, Foster City, CA, USA), while STAT3 mRNA expression was examined using SYBR Green Real-time PCR Master Mix (TaKaRa). Quantitative real-time (qRT) PCR reaction was conducted on GeneAmp PCR 9700 Thermo cycler (Applied Biosystems). Expressions of miR-506 and STAT3 mRNA were evaluated using 2−ΔΔCt method.
9
Quantitative miRNA Expression Analysis
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TaqMan miRNA assay was performed as previously described (Chen et al., 2005 (link)). Reverse transcription (RT) reactions were run in a GeneAmp PCR 9,700 Thermocycler (Applied Biosystems, USA). RT reaction without templates or primer was used as the negative controls. Gene expression levels were quantified using the ABI 7300 RT-PCR System (Applied Biosytems). A comparative real-time PCR including no template control was performed in triplicate using each specific primer set for miR-93, miR-106b, miR-18a, miR-181a, miR-199a, miR-10b, mir-126, miR-181b, miR-301, miR-127, mir-210, mir-376a, and snoRNA234 as the loading control. The reaction was performed at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. All reagents and protocols were from Applied Biosystems (USA). The expression level of each miRNA relative to snoRNA234 was determined using the 2−ΔΔCt method (Livak and Schmittgen, 2001 (link)).
10
Confirming Inverted Repeats in Plastid Genome
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PCR amplification and sequencing of the amplicons were used to confirm the inverted repeats in the plastid genome. Two pairs of primers were used to amplify the regions that join the inverted repeat to the main body of the apicoplast. The primer sets were F1: 5′-AGT CGC TAA GTA GCC AAG TTT-3′ and R1: 5′-TTG TCT TGC CTG TGC TAT AGT AAT-3′, and F2: 5′- ACT ACA TCA ACG GCT AAC T-3′ and R2: 5′-GTA CGA GAG GAC CAA AGA AA-3′, which targeted a 634 (between rps4 and LSU rRNA genes) and 743 bp fragment (between ycf24 and LSU rRNA genes), respectively. The reactions contained 1 μl of DNA, 1× GeneAmp PCR buffer (Applied Biosystems, Foster City, CA), 1.5U of GoTaq polymerase (Promega, Madison, WI), 250 μM dNTPs (Promega), 3 mM MgCl2, 250 nM of each primer, and 400 ng/μl of non-acetylated bovine serum albumin (Sigma-Aldrich, St. Louis, MO). The amplification was performed on a GeneAmp PCR 9700 thermocycler (Applied Biosystems), consisting of an initial denaturation at 94 °C for 5 min; 35 cycles at 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 1 min; and a final extension at 72 °C for 7 min. The secondary PCR products were detected by 1.5 % agarose gel electrophoresis, and sequenced in both directions using the BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI 3130xl Genetic Analyzer (Applied Biosystems).
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