RNA was extracted with TRIzol (Invitrogen) and cDNA was synthesised according to the method described in the HiScript First Strand cDNA synthesis kit (Vazyme Biotech, Nanjing, China). The quantitative reverse transcriptase (qRT)-PCR analysis was performed according to the procedure described in the UltraSYBR one-step qRT-PCR kit (CWBIO, Beijing, China). The primers used for qRT-PCR analysis are listed in supplementary table S1 .
Ultrasybr one step qrt pcr kit
Manufactured by CWBIO
Sourced in China, United States
The UltraSYBR One Step qRT-PCR Kit is a reagent kit designed for real-time quantitative reverse transcription PCR (qRT-PCR) experiments. The kit includes all the necessary components for performing one-step RT-PCR reactions, including a reverse transcriptase enzyme, a hot-start DNA polymerase, and a SYBR Green-based fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Hifi mmlv cdna kit, by CWBIO (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Hiscript first strand cdna synthesis kit, by Vazyme (1 mentions)
Nanodrop 2000 micro spectrophotometer, by Agilent Technologies (1 mentions)
Nanodrop machine, by Agilent Technologies (1 mentions)
Superrt cdna synthesis kit, by CWBIO (1 mentions)
Abi 7500 sequencing detection system, by Thermo Fisher Scientific (1 mentions)
4 protocols using ultrasybr one step qrt pcr kit
1
qRT-PCR Analysis of Gene Expression
2
qRT-PCR Analysis of hTERT Expression
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Total RNA was isolated from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentration and quality of RNA were determined using an Agilent 2100 Analyzer and a NanoDrop
2000 microspectrophotometer. Complementary DNA was reverse transcribed from total RNA using a HiFi-MmlV cDNA kit (CWBIO, Jiangsu, China), and qRT-PCR for gene expression was performed on an
ABI 7500 Sequencing Detection System (Applied Biosystems, Carlsbad, CA, USA) with the UltraSYBR One Step qRT-PCR Kit (CWBIO, Jiangsu, China). The specific primers were designed as follows:
5’-TATGCCGTGGTCCAGAAGG -3’ (hTERT, sense), 5’-CAAGAAATCATCCACCAAACG -3’ (hTERT, antisense), 5’-CGGCACAGTCAAGGCAGAGAAC -3’ (GAPDH, sense), and 5’-CCACATACTCAGCACCAGCATCAC -3’ (GAPDH,
antisense).
2000 microspectrophotometer. Complementary DNA was reverse transcribed from total RNA using a HiFi-MmlV cDNA kit (CWBIO, Jiangsu, China), and qRT-PCR for gene expression was performed on an
ABI 7500 Sequencing Detection System (Applied Biosystems, Carlsbad, CA, USA) with the UltraSYBR One Step qRT-PCR Kit (CWBIO, Jiangsu, China). The specific primers were designed as follows:
5’-TATGCCGTGGTCCAGAAGG -3’ (hTERT, sense), 5’-CAAGAAATCATCCACCAAACG -3’ (hTERT, antisense), 5’-CGGCACAGTCAAGGCAGAGAAC -3’ (GAPDH, sense), and 5’-CCACATACTCAGCACCAGCATCAC -3’ (GAPDH,
antisense).
3
Screening Target Genes in OSCC Cells
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To identify a target gene of interest for subsequent experiments, we used qRT-PCR technology to screen the expression of 14 target genes in OSCC cells. These tumor-targeted genes were obtained by analysis of gene expression pro ling database through Oncomine™ Platform (http://www.oncomine.org/resource/login.html), including ABCA1, ABCA5, ABCB6, ABCC3, ABCC4, ABCC5, ABCC10, CD20, CD22, CD47, ERBB2, PIGR, GPNMB and NECTIN4 (Table 1). RNA was isolated from OSCC cells lysates using the Trizol method (Thermo Fisher Scienti c, Waltham, MA, USA) after cells were incubated in a humidi ed atmosphere with 5% CO 2 at 37 °C, and total RNA samples were quanti ed with the Nanodrop machine (Agilent Technologies, Santa Clara, CA, USA). Next, the cDNA was generated with SuperRT cDNA Synthesis Kit (CWbiotech, Beijing, China), and UltraSYBR One Step qRT-PCR Kit (Low ROX, CWbiotech, Beijing, China) was then used to quantify the expression of a target gene.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene for normalization as previously described [21] , and gene expression was determined by the 2 -△△CT method. qRT-PCR primer sequences are listed in Table 1.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene for normalization as previously described [21] , and gene expression was determined by the 2 -△△CT method. qRT-PCR primer sequences are listed in Table 1.
4
Quantifying hTERT Gene Expression
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Total RNA was isolated from the cells with TRIzol reagent (Invitrogen, USA). The concentration and quality of RNA was determined using an Agilent 2100 Analyzer and an NanoDrop 2000 microspectrophotometer. Complementary DNA was reversely transcribed from total RNA using HiFi-MmlV cDNA kit (CWBIO, Jiangsu, China) and qRT-PCR for gene expression was performed on an ABI 7500 Sequencing Detection System (Applied Biosystems, USA) with the UltraSYBR One Step qRT-PCR Kit (CWBIO, Jiangsu, China). The speci c primers were designed as follows: 5'-TATGCCGTGGTCCAGAAGG -3' (hTERT, sense), 5'-CAAGAAATCATCCACCAAACG - 3' (hTERT, antisense); 5'-CGGCACAGTCAAGGCAGAGAAC - 3' (GAPDH, sense), and 5'-CCACATACTCAGCACCAGCATCAC - 3' (GAPDH, antisense).
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