Total RNA was obtained using an RNA extraction kit (#RN001, ES Science, Shanghai, China) and reverse-transcribed to cDNA according to the manufacturer’s instructions (#RR036A-1, TAKARA, Japan). The relative expression of the identified genes was determined and measured by quantitative RT-PCR assays (ABI Q6 System, Applied Biosystems). The expression levels of all the targeted genes were normalized to those of β-actin, and the fold changes were calculated via the 2−ΔΔCt comparative method. The sequences of the specific primers used are shown in Table S2 .
Abi q6 system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Q6 system is a real-time PCR instrument designed for nucleic acid quantification and analysis. It provides high-performance data acquisition and analysis capabilities for a variety of applications in life science research and clinical diagnostics.
Automatically generated - may contain errors
5 protocols using abi q6 system
1
Quantitative RT-PCR Gene Expression Analysis
2
Genomic DNA Isolation and SNP Genotyping
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Venous blood samples were collected from all of the study subjects via venipuncture. Genomic DNA was isolated from the peripheral blood of controls and patients using an organic phenol–chloroform procedure, and the samples were stored at −80° for future analysis [34 ]. The rs2292832 in MIR149, rs3746444 in MIR499, rs11614913 in MIR196, rs1044165 in MIR223, and rs767649 in MIR155 were genotyped using a TaqMan SNP genotyping assay. DNA amplification was performed in 384-well plates using an ABI Q6 system (Applied Biosystems, Waltham, MA, USA).
3
Genotyping miRNA-149 rs2292832 Polymorphism
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A TIANamp Blood DNA Kit (Centrifugal column, Tiangen) was used to extract DNA from the blood samples according to the manufacturer's instructions.34 , 35 , 36 TaqMan real‐time PCR assays were set up in 384‐well plates containing positive and negative samples and were carried out in an ABI Q6 system (Applied Biosystems) to genotype the miRNA‐149 rs2292832 T>C polymorphism.37 , 38 , 39
4
Quantitative Analysis of mRNA and miRNA
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Total RNA from the cultured cells was extracted using TRIzol reagent according to
the manufacturer’s instructions (Invitrogen). cDNA was synthesized using an
M-MLV reverse transcription kit (Invitrogen). MiRNA was extracted using an
RNeasy Mini Kit (Tiangen, Beijing, China) and reversed-transcribed into cDNA
using a poly (A) kit (Tiangen). qPCR analysis was performed using SYBR Green PCR
mix (Tiangen) and the ABI Q6 system (Applied Biosystems, Thermo Fisher
Scientific). All reactions were performed in triplicate. The gene-specific
primers sequences are as follows: miR-28-5p sense,
5′-AACACGCAAGGAGCTCACAG-3′; U6 sense, 5′-AACAAGCCCTGC
GCAAGGATGA-3′; BECN1 sense, 5′-GGAAGTTTTCCGG CGGCT-3′;
BECN1 antisense, 5′-AGACCCTTCCATCCCTCAGC-3′;
β-actin sense, 5′-GTCATTCCAAATATGA GATGCGT-3′; and
β-actin antisense, 5′-GCTATCACCTCCCCTGTGTG-3′. The primers
were synthesized by Invitrogen (Shanghai, China). β-actin and U6 were used as
internal control genes for the relative quantities of mRNA and miRNA,
respectively. The 2−△△Ct method was used to analyze the qPCR results.19 (link)
the manufacturer’s instructions (Invitrogen). cDNA was synthesized using an
M-MLV reverse transcription kit (Invitrogen). MiRNA was extracted using an
RNeasy Mini Kit (Tiangen, Beijing, China) and reversed-transcribed into cDNA
using a poly (A) kit (Tiangen). qPCR analysis was performed using SYBR Green PCR
mix (Tiangen) and the ABI Q6 system (Applied Biosystems, Thermo Fisher
Scientific). All reactions were performed in triplicate. The gene-specific
primers sequences are as follows: miR-28-5p sense,
5′-AACACGCAAGGAGCTCACAG-3′; U6 sense, 5′-AACAAGCCCTGC
GCAAGGATGA-3′; BECN1 sense, 5′-GGAAGTTTTCCGG CGGCT-3′;
BECN1 antisense, 5′-AGACCCTTCCATCCCTCAGC-3′;
β-actin sense, 5′-GTCATTCCAAATATGA GATGCGT-3′; and
β-actin antisense, 5′-GCTATCACCTCCCCTGTGTG-3′. The primers
were synthesized by Invitrogen (Shanghai, China). β-actin and U6 were used as
internal control genes for the relative quantities of mRNA and miRNA,
respectively. The 2−△△Ct method was used to analyze the qPCR results.19 (link)
5
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was obtained using an RNA extraction kit (#RN001; ESscience, Shanghai, China) and converted to cDNA according to the manufacturer's instructions (#RR036A‐1; TAKARA, Japan). The relative expressions of identified genes were performed and measured by quantitative real‐time PCR assays (ABI Q6 System, Applied Biosystems). All the genes were normalized to GAPDH, and the fold change was calculated as 2–ΔΔCT comparative thresholds. Specific primer sequences are shown in Table S1 .
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