The harvested cells were dissolved in TRIzol® reagent (Invitrogen, USA), and total mRNA was then extracted following the manufacturer's protocol. qRT-PCR was performed using SYBR Green PCR Mix (Tiangen, China) and a Stratagene Mx3005P sequence detection system (StrataGene, Agilent, USA). The amplification parameters were as follows: 95 °C for 15 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. The 2−∆∆Ct method was performed with the GAPDH gene as an internal control, and the relative quantification procedure was selected. The primer sequences are listed in Table s1 .
Sybr green pcr mix
Manufactured by Tiangen Biotech
Sourced in China
SYBR Green PCR Mix is a ready-to-use solution designed for real-time quantitative PCR (qPCR) analysis. It contains SYBR Green I dye, DNA polymerase, dNTPs, and necessary buffers for efficient DNA amplification and detection.
Automatically generated - may contain errors
4 protocols using sybr green pcr mix
1
Quantitative RT-PCR Gene Expression Analysis
2
Quantitative Analysis of RNA Expression
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Total RNA of cells or tissues was isolated using the Trizol reagent (Qiagen, Hilden, Germany). The expression levels of LUADT1, miR-1207-5p and SEPT9 were detected with the SYBR green PCR mix (TIANGEN, Beijing, China) using the 7500 Fast PCR platform following the protocol of 95°C for 55 s, 42 cycles at 95°C for 15 s, and 60°C for 40 s.
GAPDH and
U6 RNA levels were also detected as the endogenous controls for
LUADT1,
SEPT9 and miR-1207-5p, respectively. The expression change of target genes was calculated according to the 2
–ΔΔCq formula. The primer sequences are as follows:
LUADT1 F, 5′-TTTCCGTTCAGCAAATCCACAC-3′ and R, 5′- TTAGGTCCAGCACTGTTATCCA-3′; miR-1207-5p F, 5′-GCCAGATCTTGATTGACTTACAGCCCAGTT-3′ and R, 5′-GCCGAATTCCACCTGTCTTTATTCCACCC-3′;
GAPDH F, 5′-GCGCCCAATACGACCAA-3′ and R, 5′-CTCTCTGCTCCTCCTGTTC-3′;
U6 RNA F, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and R, 5′-CGCTTCACGAATTTGCGT-3′; and
SEPT9 F, 5′-ACCGGATCTCAGCCTTGAA-3′ and R, 5′-GAGAGCTCCACCCTCCGCAGG-3′.
GAPDH and
U6 RNA levels were also detected as the endogenous controls for
LUADT1,
SEPT9 and miR-1207-5p, respectively. The expression change of target genes was calculated according to the 2
–ΔΔCq formula. The primer sequences are as follows:
LUADT1 F, 5′-TTTCCGTTCAGCAAATCCACAC-3′ and R, 5′- TTAGGTCCAGCACTGTTATCCA-3′; miR-1207-5p F, 5′-GCCAGATCTTGATTGACTTACAGCCCAGTT-3′ and R, 5′-GCCGAATTCCACCTGTCTTTATTCCACCC-3′;
GAPDH F, 5′-GCGCCCAATACGACCAA-3′ and R, 5′-CTCTCTGCTCCTCCTGTTC-3′;
U6 RNA F, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and R, 5′-CGCTTCACGAATTTGCGT-3′; and
SEPT9 F, 5′-ACCGGATCTCAGCCTTGAA-3′ and R, 5′-GAGAGCTCCACCCTCCGCAGG-3′.
3
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)
4
Quantitative RT-PCR Gene Expression Analysis
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The harvested cells were dissolved in TRIzol® reagent (Invitrogen, USA), and total mRNA was then extracted following the manufacturer's protocol. QRT-PCR was performed using SYBR Green PCR Mix (Tiangen, China) and a Stratagene Mx3005P sequence detection system (StrataGene, Agilent, USA). The ampli cation parameters were as follows: 95 °C for 15 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. The 2-∆∆Ct method was performed with the GAPDH gene as an internal control, and the relative quanti cation procedure was selected. The primer sequences are listed in Table S2.
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