Total RNA was extracted from untreated WM-266-4 cells and WM 266-4 cells treated with the IC50 of ORT, DTIC, or the ORT/DTIC combination, using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Complementary DNA (cDNA) was synthesized from total RNA using a first-strand synthesis kit (GeneAll). The cDNA concentration was determined using a NanoDrop spectrophotometer (Coliri LB 915; JC Bio). qRT-PCR was performed using CFX Connect Real-Time PCR (Bio-Rad) and BrightGreen 2X qPCR MasterMix (ABM, Vancouver, Canada), with 50 amplification cycles. In the present study, the expression levels of NOTCH2, NOTCH3, MYC, CCND1, BAX, CASP3 and CASP9 were comparatively analyzed among different treatment groups. The primers used in the qRT-PCR analysis are listed in Table I . The expression levels of target genes were normalized to those of GAPDH (19 (link)–24 (link)). Relative gene expression levels were calculated using the 2−ΔΔCt method (25 (link)).
First strand synthesis kit
Manufactured by GeneAll
The First-strand synthesis kit is a laboratory tool used to produce complementary DNA (cDNA) from RNA templates. It contains the necessary reagents and enzymes to perform the reverse transcription process, which is a key step in many molecular biology and genomics applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cfx connect real time pcr, by Bio-Rad (1 mentions)
Brightgreen 2x qpcr mastermix, by Applied Biological Materials (1 mentions)
Oligo dt, by GeneAll (1 mentions)
Sv total rna isolation system, by Promega (1 mentions)
Quantity one 1 d analysis software, by Bio-Rad (1 mentions)
2 protocols using first strand synthesis kit
1
Gene Expression Analysis of Melanoma Cells
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated with SV Total RNA Isolation System (Promega). RNA concentration was evaluated by spectrophotometric reading at 280 and 260 nm. Total RNA was used for first strand cDNA synthesis with HyperScript, First strand Synthesis Kit and Oligo-dT, as random primer (GeneAll). The PCR was performed with about 150 ng of cDNA using DreamTaq. The following primer sequences were used for amplification: Actin forward 5'-CCTTCCTGGGCATGGAGTCCTG-3', Actin reverse 5'-GGAGCAATGATCTTGATCTTC-3' (208 bp); Bax forward 5'-GCAGGGAGGATGGCTGGGGAG-3', Bax reverse 5'-TCCAGACAAGCAGCCGCTCACG-3' (352 bp).
The experimental protocols for PCR reactions were: initial denaturation for 5 min at 95°C; amplification for 40 cycles of denaturation, 30 sec, 95°C, annealing: 30 s, at 55°C (Actin), 60°C (Bax) and elongation at 72°C for 1 min; final elongation for 10 min at 72°C. PCR products were then analysed by 1.5 % agarose gels electrophoresis in TBE1X Buffer. Image acquisition and product analysis was made by Bio-Rad imaging systems with Quantity One1-D analysis software. The density of the PCR bands were divided by that of the housekeeping gene (Actin) and expressed as percent of the control band density.
The experimental protocols for PCR reactions were: initial denaturation for 5 min at 95°C; amplification for 40 cycles of denaturation, 30 sec, 95°C, annealing: 30 s, at 55°C (Actin), 60°C (Bax) and elongation at 72°C for 1 min; final elongation for 10 min at 72°C. PCR products were then analysed by 1.5 % agarose gels electrophoresis in TBE1X Buffer. Image acquisition and product analysis was made by Bio-Rad imaging systems with Quantity One1-D analysis software. The density of the PCR bands were divided by that of the housekeeping gene (Actin) and expressed as percent of the control band density.
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