Total RNA was extracted from tissues or cells with the TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA) and then reversely transcribed into cDNA using the Primer Script Kit (TaKaRa, Dalian, P.R. China). RT-PCR was performed with a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) under the following conditions: 95°C for 30 s, 35 cycles of 95°C for 15 s and 60°C for 30 s. The PCR primers were as follows: USP22, 5′-CCATTGATCTGATGTACGGAGG-3′ (forward) and 5′-TCCTTGGCGATTATTTCCATGTC-3′ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GAGTCAACGGATTTGGTCGT-3′ (forward) and 5′-GACAAGCTTCCCGTTCTCAG-3′ (reverse). GAPDH was used as an internal control. Data analysis was performed through the 2−ΔΔCT method21 (link).
Primer script kit
Manufactured by Takara Bio
Sourced in China
The Primer Script Kit is a laboratory product designed for the reverse transcription of RNA into complementary DNA (cDNA). The kit includes all the necessary components for the efficient conversion of RNA into a cDNA template, which can then be used for various downstream applications, such as polymerase chain reaction (PCR) and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green, by Takara Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Merck Group (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green reaction mix, by Vazyme (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Trizol, by Beyotime (1 mentions)
6 protocols using primer script kit
1
Quantitative RT-PCR for USP22 expression
2
Quantifying Gene Expression Profiles in Cells
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Trizol reagent was used to extract total cellular RNA from tissues and cells. First strand cDNA was synthesized with the PrimerScript kit (Takara, Dalian, Liaoning, China). RT-qPCR was performed using SYBR Green (Takara) at the Applied Biosystems 7500 (Foster City, CA, United States). The Delta Delta Ct (ddCt) method was used to measure relative gene expression levels. Primers were as follows: SNHG17: 5'-GTTCCTGGGGCTTGGATGAT-3' (sense), 5'-GATCTAAGGCTGAGACCCACG-3' (antisense); STC2: 5'-TCTTGTGAGATTCGGGGCTT-3' (sense), 5'-ACAGGTCGTGCTTGAGGTAG-3' (antisense); ki-67: 5'-GAGGTGTGCAGAAAATCCAAA-3' (sense), 5'-CTGTCCCTATGACTTCTGGTTGT-3' (antisense); Bcl-2: 5'-CGACTTTGCAGAGATGTCCA-3' (sense), 5'-ATGCCGGTTCAGGTACTCAG-3' (antisense); Bax: 5'-TGCAGAGGATGATTGCTGAC-3' (sense), 5'-GAGGACTCCAGCCACAAAGA-3' (antisense); GAPDH: 5'-GTCTCCTCTGACTTCAACAGCG-3' (sense), 5'-ACCACCCTGTTGCTGTAGCCAA-3' (antisense); miR-361-3p: 5'-TCCCCCAGGTGTGATTCTGATTT-3' (sense), 5'-GCAAATCAGAATCACACCTG-3' (antisense); U6 snRNA: 5'-CTCGCTTCGGCAGCACA-3' (sense), 5'-AACGCTTCACGAATTTGCGT-3' (antisense). The RT-qPCR procedure used was: 95°C for 5 min, followed by 30 cycles at 95°C for 10s and 60°C for 30s.
3
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from MDA-MB-231 in the co-culture system using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After reverse transcription, cDNA was amplified with 1 μg of total RNA using a Primer Script Kit (TaKaRa, Dalian, China). The mRNA expression of target genes was normalized to that of β-actin. Primers used in this study are shown in Table 1 .
4
RNA Extraction and RT-qPCR Protocol
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RNA extraction and RT-qPCR were performed as described previously [20 ]. Briefly, total RNA was extracted using TRIzol Reagent (Takara, Japan). After quantitation, 1 μg RNA was reverse transcribed to cDNA using a PrimerScript kit (Takara, Japan). The qPCR was performed using a SYBR Green reaction mix (Vazyme, China). Relative expression of interested genes was measured using the 2−ΔΔCt method.
5
Quantification of miRNA and mRNA Levels
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RNA samples were isolated by Trizol (Beyotime, Haimen, Jiangsu, China) according to the provided protocols. After concentration quantification using NanoDrop-1000, RNA was reverse transcribed into complementary DNA using PrimerScript kit (Takara, Dalian, Liaoning, China). RT-qPCR was performed at QuanStudio 6 (Invitrogen) using SYBR Green (Takara). Primers were: miR-663a: forward, 5’-GTGCGTGTCGTGGAGTCG-3’ and reverse, 5’-TTTAGGCGGGGCG-3’; U6 snRNA forward, 5’-GCTTCGGCAGCACATATACTAAAAT-3’ and reverse, 5’-CGCTTCACGAATTTGCGTGTCAT-3’; FHL3: forward, 5′-CATGGCATGAGCACTGCTTCCTG-3′ and reverse, 5′-GCTTAGGGCCCTGCCTGGCTACAGC-3′; GAPDH: forward, 5′-GCACCGTCAAGGCTGAGAAC-3′, reverse, 5′-TGGTGAAGACGCCAGTGGA-3′. Comparative Ct method was performed to calculate relative gene expression level.
6
Quantitative RNA Expression Analysis
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Total RNA extraction was performed using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was amplified with 1 µg of total RNA using a Primer Script Kit (TaKaRa, Dalian, China). Primers used for RT-PCR of BMP9 and HER2 are shown in Table 1 . The mRNA expression of target genes was normalized to those of GAPDH.
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