Total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific Inc.). RNA was reverse‐transcribed to complementary DNA using a PrimeScript RT Reagent Kit (Takara Biotechnology Co., Ltd., Dalian, China). Quantitative RT‐PCR was performed using SYBR Premix Real‐time PCR Reagent (Takara Biotechnology) on an ABI 7500HT instrument (Bio‐Rad, Hercules, CA). For mRNA detection, the primers used in this study were as follows: AOC3 (forward: 5′-TACAACCACTGCACTACCTG-3′, reverse: 5′-TGGAATGCTTGAAGGCTGCT-3′) and GAPDH as an internal control (forward: 5′-ACCTGACCTGCCGTCTAGAA-3′, reverse: 5′-TCCACCACCCTGTTGCTGTA-3′). Each experiment was performed in triplicate, and the data were analyzed by the 2−△△Ct method.
Sybr premix real time pcr reagent
Manufactured by Takara Bio
Sourced in United States
SYBR Premix Real-time PCR Reagent is a ready-to-use solution for real-time PCR amplification and detection using SYBR Green dye. It contains all the necessary components, including DNA polymerase, dNTPs, and buffer, to perform real-time PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Hairpin it microrna and u6 snrna normalization rt pcr quantitation kit, by GenePharma (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Rnaiso plus kit, by Takara Bio (1 mentions)
Lightcycler 480 2 system, by Roche (1 mentions)
Dual beam ultraviolet spectrophotometer, by Eppendorf (1 mentions)
Abi 7900 real time pcr system, by Thermo Fisher Scientific (1 mentions)
6 protocols using sybr premix real time pcr reagent
1
Quantitative RT-PCR for mRNA Detection
2
Quantitative Real-Time PCR Analysis of Ep3 Expression
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Total mRNA was extracted from tissues and cells using TRIzol® (Thermo Fisher Scientific, Inc.) according to the manufacturers' protocol. mRNA was reverse transcribed to cDNA using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturers' protocol. The mRNA levels of Ep3 and β-actin were measured using SYBR Premix Real-Time PCR reagent (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. The primers used in the present study were as follows: Ep3 forward, 5′-TCCTTCCTAATCGCCGTTC-3′ and reverse, 5′-CTCCGCTTCAGGTTGTTCAT-3′; β-actin forward, 5′-CCTGGACTTCGAGCAAGAGA-3′ and reverse, 5′-ACTTGCGCTCAGGAGGAGCA-3′. The PCR conditions were as follows: 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C, and a final extension for 5 min at 72°C. The relative expression level of Ep3 was normalized to β-actin levels. The relative expression levels were calculated using the 2−ΔΔCt method (30 (link)).
3
Quantification of miR-613 and TAGLN2 expression
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Total RNA extraction from cells and tissues was performed using the miRNeasy Mini Kit (Qiagen, Germantown, MD, USA), and non-coding small RNA reverse transcription was performed using the Hairpin-it™ microRNA and U6 snRNA Normalization RT-PCR Quantitation Kit (GenePharma, Shanghai, China). Complementary cDNA was obtained using the PrimeScript RT Reagent Kit (Takara, Dalian, China). Further, qRT-PCR analyses for the detection of miR-613 and TAGLN2 were conducted using SYBR Premix Real-time PCR Reagent (Takara) and the ABI 7500 FAST Real Time PCR System (Applied Biosystems, Foster City, CA, USA). All the primers were purchased from GenePharma. The primer sequences were as follows: miR-613, forward primer 5′-CGGCCGCGAGGAATGTTCCTTC-3′, reverse primer 5′-ATCCAGTGCAGGGTCCGAGG-3′; and TAGLN2, forward primer 5′-ATCACCACCCAGTGCCGAAAG-3′, reverse primer 5′-CATGGTGGAGGCCTGGATCTT-3′. Further, the fold-change in the expression of each gene was determined using the 2−ΔΔCt method. U6 and GAPDH were chosen as reference genes for miR-613 and TAGLN2, respectively.
4
Quantitative PCR Analysis of Gene Expression
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Total RNA was extracted using an RNAiso Plus Kit (Takara, Dalian, China) according to the manufacturer’s instructions. After reverse transcription, cDNA was synthesized, and qPCR analysis was performed using SYBR Premix Real-time PCR Reagent (Takara) in a Roche LightCycler 480 II system (Roche Diagnostics Corporation, Indianapolis, IN, USA) according to the manufacturer’s protocol. The internal control was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences of the selected target genes and internal controls are listed in Table 2 . The target gene expression levels were calculated using the 2 –ΔΔCt method, similar to that in one of our previously reported studies [24 (link)].
Primer sequences of genes used in this study
| Gene | Primer sequence | Product length (bp) |
|---|---|---|
| FDX1 | Forward: 5′-CTTTGGTGCATGTGAGGGAA-3′ Reverse: 5′-GCATCAGCCACTGTTTCAGG-3′ | 216 |
| P2RX4 | Forward: 5′-TGGCGGATTATGTGATACCAGC-3′ Reverse: 5′-GTCGCATCTGGAATCTCGGG-3′ | 112 |
| CDK1 | Forward: 5′-CCCTTTAGCGCGGATCTACC-3′ Reverse: 5′-CATGGCTACCACTTGACCTGT-3′ | 133 |
| MAP1LC3A | Forward: 5′-CCAGCAAAATCCCGGTGA-3′ Reverse: 5′-TGGTCCGGGACCAAAAACT-3′ | 88 |
| GAPDH | Forward: 5′-GCACCGTCAAGGCTGAGAAC-3′ Reverse: 5′-TGGTGAAGACGCCAGTGGA-3′ | 138 |
5
Quantification of miR-107 and BDNF expression
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Total RNA of the cultured cells and the tissues was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. The purity and concentration of total RNA were determined by a dual-beam ultraviolet spectrophotometer (Eppendorf, Hamburg, Germany). Then, a total of 3 μg of mRNA was reverse transcribed to single-stranded cDNAs using a PrimeScript® RT reagent kit (Takara Biotechnology Co., Ltd., Dalin, China). qRT-PCR for miR-107 and BNDF were performed using SYBR premix real-time PCR Reagent (Takara) under an ABI7900 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primers for miR-107 and U6 were brought from Applied Biosystems. The primers for BDNF and β-actin used in this study were as described previously (20 (link)). U6 RNA was used to normalize the miR-107 RNA levels, and β-actin was used to normalize the level of BDNF mRNAs. The comparative 2−ΔΔCt method was employed for relative quantification.
6
Quantifying miR-223 and STIM1 in Breast Cancer
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According to the manufacturer's instructions, total RNA was extracted from each groups of breast cancer cells using Trizol (Invitrogen ‚ USA) and subsequently reverse transcribed to cDNA using a reverse-transcription PCR system (TaKaRa).The expression levels of miR-223 and STIM1 mRNA were analyzed by using SYBR premix real-time PCR Reagent (TaKaRa). The GAPDH gene was used as an internal control. The primer sequences are shown as follows: miR-223(forward: 5′-GCGTGTATTTGACAAGCTGAGTT-3 and reverse: 5′-GTGTCAGTTTGTCAAATACCCCA-3); STIM1(forward: 5'-TGTGTCTCCCTTGTCCATGC-3' and reverse, 5'-CATCTGAGGTTTGGGGG-3').
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