Total RNA was extracted using Trizol (TransGen Biotech Co, Ltd). RNA (2.5 μg) was reverse-transcribed using the EasyScipt First-strand cDNA Synthesis SuperMix (TransGen Biotech), according to the manufacturer’s instructions. PCR amplification was performed using the following primers: for USP7, Forward 5′-TTTTGTGCGAAATCTGCC-3′ and Reverse 5′-AATCCCACGCAACTCCAT-3′; for RNF6, Forward 5′-CATCAGTGGCTCTTCGGTCA-3′ and Reverse5′-ATGCTCATAGTGCCTGGTGG-3′; for GAPDH, Forward 5′-AGTCCACTGGCGTCTTCA-3′ and Reverse 5′-CTCCGACGCCTGCTTCACCA-3′. Reaction cycling conditions were 3 min at 95 °C, followed by 30 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 40 s, and 1 cycle at 72 °C for 10 min. Products were analyzed on 2% agarose gels (TransGen Biotech). The PCR products were visualized by Goldview staining (TransGen Biotech) following electrophoresis on 2% agarose gels.
Goldview staining
Manufactured by Transgene
Sourced in China
Goldview staining is a laboratory equipment product that is used for the visualization of nucleic acids, such as DNA and RNA, in agarose gels. It is a fluorescent dye that binds to nucleic acids and emits a bright, yellow-gold color when exposed to ultraviolet (UV) light. The product is designed to provide a simple and efficient way to detect and analyze nucleic acid samples in a laboratory setting.
Automatically generated - may contain errors
6 protocols using goldview staining
1
Quantitative RT-PCR Analysis of USP7, RNF6, and GAPDH
2
RT-PCR Analysis of Gene Expression
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The RT-PCR was performed as described previously15 (link). The primers for Otub1 were 5′-TGTTTCTATCGGGCTTTCG-3′ (forward) and 5′-AGATGTGCGGATTGGTGG-3′ (reverse). The specific primers for c-Maf, GAPDH, CCND2, and ITGB7 were described previously15 (link). PCR products were visualized by GoldView® staining (TransGen Biotech Co., Ltd., Beijing, China) following electrophoresis on 2% agarose gels.
3
Analysis of Gene Expression
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Total RNA was extracted using Trizol (Sangon Biotech, Shanghai, China). Total RNA (2 µg) was reverse transcribed using an EasyScipt First-Strand cDNA Synthesis kit (Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. PCR amplification was carried out using the following primers: USP5, 5’-CGG ATT TGA CCT TAG CG-3’ (forward) and 5’-CTG CCA TCG AAG TAG CG-3’ (reverse); c-Maf, 5’-AAA AAG GAA CCG GTG GAG AC-3’ (forward) and 5’-GGT AGC CGG TCA TCC AGT AG-3’ (reverse); and GAPDH, 5’-AAT CCC ATC ACC ATC TTC C-3’ (forward) and 5’-CAT CAC GCC ACA GTT TCC-3’ (reverse). The primers used for CCND2, ITGB7, and ARK5 were described previously [11 (link)]. PCR products were visualized by GoldView® staining (TransGen Biotech Co., Ltd., Beijing, China) following electrophoresis on 2% agarose gels.
4
Quantitative PCR Analysis of Genetic Targets
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Total RNA was extracted using Trizol® (Transgene, Beijing, China). RNA (2.5 μg) was reverse-transcribed using an EasyScipt® First-strand cDNA Synthesis SuperMix® (Transgen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instruction. PCR amplification was performed using the following primers: for USP7, 5′-TTTTGTGCGAAATCTGCC-3′ (F) and 5′-AATCCCACGCAACTCCAT-3′ (R); for BCR-ABL, 5′-GAAGAAGTGTTTCAGAAGCTTCTCCC-3′ (F) and 5′-GACCCGGAGCTTTTCACCTTTAGTT-3′ (R); for GAPDH, 5′-AATCCCATCACCATCTTCC-3′ (F) and 5′-CATCACGCCACAGTTTCC-3′ (R). The PCR was run as described previously22 (link). The PCR products were visualized by Goldview staining (Transgen) following electrophoresis on 2% agarose gels.
5
RNA Extraction and RT-PCR Analysis
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Total RNA was extracted using Trizol (Sangon Biotech, Shanghai, China). RNA (2 μg) was reverse-transcribed using an EasyScipt First-strand cDNA Synthesis (Vanzyme, Nanjing, China) according to the manufacturer’s instruction. PCR amplification was carried out using the following primers: for USP5, 5′-CGGATTTGACCTTAGCG-3′ (Forward) and 5′-CTGCCATCGAAGTAGCG-3′ (Reverse); for GAPDH, 5′-AATCCCATCACCATCTTCC-3′ (Forward) and 5′-CATCACGCCACAGTTTCC-3′ (Reverse); for CCND2, 5′-ATTTACACCGACAACTCCATC-3′ (Forward) and 5′-CTCAGTCAGGGCATCACAA-3′ (Reverse); for ITGB7, 5′-GACGCCAAGATCCCATCC-3′ (Forward) and 5′-GGGTATCCCTCAGCACGAA-3′ (Reverse); for ARK5, 5′-GTCCTGCCTTACCCTCTACT-3′ (Forward) and 5′-CAGGCTCTGACAGGGATT-3′ (Reverse). The PCR products were visualized by Goldview staining (Transgen, Beijing, China), following electrophoresis on 2% agarose gels.
6
RNA Extraction and Gene Expression Analysis
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Total RNA was extracted using Trizol (Sangon Biotech). RNA (1 μg) was reverse transcribed using an EasyScript First-strand cDNA Synthesis (Vazyme) according to the manufacturer's instruction. PCR amplification was carried out using the following primers: for USP10, 5′-AATAAAGGGAACTGGTGC-3′ (Forward) and 5′-CTATCATGGGTGTTGACGT-3′ (Reverse); for GAPDH, 5′-AATCCCATCACCATCTTCC-3′ (Forward) and 5′-CATCACGCCACAGTTTCC-3′ (Reverse); for PTEN, 5′-ACCATAACCCACCACAGC-3′ (Forward) and 5′-ACCAGTTCGTCCCTTTCC-3′ (Reverse). The PCR products were visualized by GoldView staining (TransGen), following electrophoresis on 1.5% agarose gels.
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