Human synovial fibroblasts (2 × 105 cells/well) in six‐well plates were incubated with the serum‐free medium containing 0, 50, 100 or 200 nm recombinant lumican for 48 h. The total RNA was extracted from the fibroblasts using the TRIzol reagent (Carlsbad,California,Invitrogen, USA), according to the manufacturer's protocol as previously described [26 ]. The first‐strand cDNA was synthesized by using the PrimeScript™ RT Master Mix Kit (Tokyo,Takara, Japan). Quantitative real‐time PCR was performed with the One Step TB Green™ PrimeScript™ RT‐PCR Kit II (SYBR Green) (Takara) on the ABI StepOnePlus real‐time PCR system (Carlsbad, California, Applied Biosystems, USA). Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used for internal control for normalization. The sequences of the primers used in this study were summarized in Table 1 .
One step tb green primescript rt pcr kit 2 sybr green
Manufactured by Takara Bio
Sourced in Japan, China
The One Step TB Green™ PrimeScript™ RT-PCR Kit II (SYBR Green) is a reagent kit designed for reverse transcription and real-time PCR amplification of RNA targets. The kit utilizes SYBR Green I dye for detection of PCR products.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Abi steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix kit, by Takara Bio (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Avantor (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Takara Bio (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cell cycle detection kit, by Keygen Biotech (1 mentions)
Annexin 5 apc 7 aad apoptosis detection kit, by Keygen Biotech (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
One step rt pcr kit, by Takara Bio (1 mentions)
Real time pcr system, by Takara Bio (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Goat anti rabbit igg hrp, by Keygen Biotech (1 mentions)
6 protocols using one step tb green primescript rt pcr kit 2 sybr green
1
Lumican Regulation of Synovial Fibroblasts
2
Anticancer Effects of Tenacigenin B
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The following materials were used in this study: tenacigenin B (HP Bio Shanghai Technology Co., Ltd.); fetal bovine serum (FBS; ExCell Biology, FSS500, USA); RPMI-1640 culture medium (Gibco, 31800-105, USA); Annexin V-APC/7-AAD apoptosis detection kit (Jiangsu Keygen Biotech Corp., Ltd., KGA1024; China); MTT (Amresco, 0793, USA); DMSO (Sigma, D2650, USA); cell cycle detection kit (Jiangsu Keygen Biotech Corp., KGA511; China); TRIzol (Invitrogen, 15596-026, USA); first-strand cDNA synthesis kit (TaKaRa, RR036B, Japan); One Step TB Green™ PrimeScript™ RT-PCR Kit II (SYBR Green) (TaKaRa, RR086B, Japan); PTEN, PI3K, AKT, p-AKT, P53, and P21 (Abcam, Cambridge, UK).
3
Quantification of Gene Expression in Cardiac Tissues
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TRIzol (15,596–026, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was utilized to extract total RNA from rat myocardial tissues or H9c2 cells in accordance with a different group (IR/HR or sham/control), and reverse transcribed 2 µg of the extracted total RNA using the 5 × PrimeScript RT Master Mix (RR036B, Takara Biotechnology, Dalian, China) per manufacturer’s instructions. The Step one plus Real time-PCR system (Applied Biosystems 7500, USA) was employed to perform qRT‐PCR assays by applying One Step TB Green™ PrimeScript™ RT-PCR Kit II (SYBR Green) (RR086B, Takara Biotechnology, Dalian, China). The specific primers are listed in Table 1 . Results were normalized by GAPDH and expressions. Each sample employed three technical replicates. The 2 −ΔΔCt method was exercised to assess gene abundance.
qRT-PCR primer sequences
| Gene | Primer sequences (5ʹ→3ʹ) |
|---|---|
| Foxp1 | F:5ʹ-TCAACCATCCAGAACGGGTC-3’ |
| R:5ʹ-ACCGAGTGTCACGAAGTGTC-3’ | |
| Pik3ip1 | F:5ʹ-GAGACCACTTCCGGTGACAAA-3’ |
| R:5ʹ-ACACGTAGCCCAAAGTTCCC-3’ | |
| GAPDH | F:5ʹ-TGGAGTCTACTGGCGTCTT-3’ |
| R:5ʹ-TGTCATATTTCTCGTGGTTCA-3’ |
4
Quantification of Autophagy-related Genes
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Total RNA, including miRNAs, was extracted from U251 cells using the TRIzol reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer's protocol. RNAs were reverse-transcribed using a cDNA First Strand cDNA Synthesis Kit(TaKaRa RR036B, Japan). For quantification, the One Step TB Green™ PrimeScript™ RT-PCR Kit II (SYBR Green) (TaKaRa RR086B, Japan) were utilized to perform qPCR following the manufacturer's instructions with a fluorescence qPCR thermal cycler (Step one plus RT-PCR system, ABI, USA). The expression of mRNA was defined from the threshold cycle (Ct), and relative expression levels were calculated using the 2−ΔΔCt method after normalization to the expression of GAPDH. RT-PCR primers are Beclin1 (Sense: 5ˊ-AATGGTGGCTTTCCTGGACT-3ˊ; Antisense: 5ˊ-TGATGGAATAGGAG CCGCCA-3ˊ), ATG5 ( Sense: 5ˊ-TGACGTTGGTAACTGACAAAG TG-3ˊ; Antisense:5ˊ-ATGCCATTTC AGTGGTGTGC-3ˊ) and GAPDH(Sense: 5ˊ-CAAATTCCA TGGCACCGTCA-3ˊ; Antisense:5ˊ-AGCATCGCCCCACTTGATTT-3ˊ).
5
Quantitative RT-PCR for Immune Markers
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Following the protocol outlined in a previous study 20 (link), total RNA was extracted from the cell lines using TRIzol reagent (15596-026, Invitrogen, USA). Total RNA (1 μg) from each sample was reverse-transcribed into cDNA using a One-Step RT-PCR kit (RR036B, TaKaRa, Japan). For real-time PCR, we employed the One-Step TB Green™ PrimeScript™ RT-PCR Kit II (SYBR Green) (RR086B, TaKaRa, Japan), and utilized an ABI Step One Plus Real-time PCR system (USA) according to the manufacturer's instructions. GAPDH was used as a housekeeping gene, and the relative expression levels of the genes were calculated using the 2-(ΔCt) method. The primers were synthesized by KeyGEN Biosynthesis (3'-5'):
CCR4 (F-CTGTGGTGGTTCTGGTCCTGTTC; R-AGATCCGAGATGGCAAGGTTGAG),
CXCR3 (F-CCTTCCTGCTCCACCTAGCTGTAG; R-GCTCCTGCGTAGAAGTTGATGTTGA),
P2RY14 (F-GGTCTCTGAAACGTGCTCTTCTAC; R-TTGCTGTAACTCACTGACTGGATGA),
CCR2 (F-CCTGAGTC) R-GAGTAGAGCGGAGGCAGGAGTT),
CCR8 (F-GGTTGGTGCTCATTGTGGTCATTG; R-GCTGTTGGCTTATGCTACATCCATC),
CCL19 (F-GCCTGCTGGTTCTCTGGACTTC; R-AGGGATGGGTTTCTGGGTCACA),
and GAPDH (F-AGATCATCAGCAATGCCTCCT; R-TGAGTCCTTCCACGATACCAA).
CCR4 (F-CTGTGGTGGTTCTGGTCCTGTTC; R-AGATCCGAGATGGCAAGGTTGAG),
CXCR3 (F-CCTTCCTGCTCCACCTAGCTGTAG; R-GCTCCTGCGTAGAAGTTGATGTTGA),
P2RY14 (F-GGTCTCTGAAACGTGCTCTTCTAC; R-TTGCTGTAACTCACTGACTGGATGA),
CCR2 (F-CCTGAGTC) R-GAGTAGAGCGGAGGCAGGAGTT),
CCR8 (F-GGTTGGTGCTCATTGTGGTCATTG; R-GCTGTTGGCTTATGCTACATCCATC),
CCL19 (F-GCCTGCTGGTTCTCTGGACTTC; R-AGGGATGGGTTTCTGGGTCACA),
and GAPDH (F-AGATCATCAGCAATGCCTCCT; R-TGAGTCCTTCCACGATACCAA).
6
HMGA2 regulation in renal cell carcinoma
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Materials: ACHN cells, a renal cell carcinoma cell line, were obtained from the Cell Bank of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College (Beijing, China). The Trizol TM reagent was obtained from ThermoFisher Scientific (Waltham, MA, USA). Rabbit anti-HMGA2 antibody (Ab207301), Rabbit anti-Ecadherin antibody (Ab40772), Rabbit anti-N-cadherin antibody (Ab76011), Rabbit anti-Snail antibody (Ab216347) were purchased from Abcam (Boston, MA, USA). Goat anti-rabbit IgG-HRP was purchased from KeyGenBioTECH (Nanjing, China). The Reverse Transcription kit and One
Step TB Green™ PrimeScript™ RT-PCR Kit II (SYBR Green) were purchased from TaKaRa-Bio (Dalian, China). The primers were ordered from Beijing Aoke Biotechnology Co., Ltd. (Beijing, China). The bicinchoninic acid (BCA) protein assay kit was purchased from Shenyang Wanlei Biological Technology Co. Ltd. (Shenyang, China). Three HMGA2 shRNAs and negative control scrambled shRNA were custom-designed and synthesized by KeyGenBioTECH (Nanjing, China).
Step TB Green™ PrimeScript™ RT-PCR Kit II (SYBR Green) were purchased from TaKaRa-Bio (Dalian, China). The primers were ordered from Beijing Aoke Biotechnology Co., Ltd. (Beijing, China). The bicinchoninic acid (BCA) protein assay kit was purchased from Shenyang Wanlei Biological Technology Co. Ltd. (Shenyang, China). Three HMGA2 shRNAs and negative control scrambled shRNA were custom-designed and synthesized by KeyGenBioTECH (Nanjing, China).
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