RNA of HeLa cells was extracted using RNAiso Plus (Takara). A reverse transcription-polymerase chain reaction kit (Takara) and SYBR Premix Ex Taq II kit (TaKaRa) were used to detect the expression of S100A16. The relative expression level of each gene was calculated using the formula: F=2−ΔΔCt. Primer sequences as follows:
Reverse transcription polymerase chain reaction kit
Manufactured by Takara Bio
Sourced in Japan
The Reverse transcription-polymerase chain reaction (RT-PCR) kit is a laboratory tool used for the detection and quantification of RNA targets. It combines the processes of reverse transcription and polymerase chain reaction to amplify and analyze specific RNA sequences.
Automatically generated - may contain errors
4 protocols using reverse transcription polymerase chain reaction kit
1
Quantifying S100A16 Expression in HeLa Cells
2
Fusion Gene Amplification from Total RNA
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cDNA synthesis from total RNA and subsequent PCR were performed using the Reverse Transcription Polymerase Chain Reaction Kit (Takara, Ohtsu, Japan) according to the manufacturer’s instructions and as previously described [14 (link)]. The sense and antisense primers used to amplify the fusion gene were 5'-CCCACTAGTTACCCACCCCA-3' (EWSR1 exon 7) and 5'-AAAACTCCACTAGGAAATCCATTT-3' (ATF exon 4), respectively. The sense and antisense primers used to amplify glycerol-3-phosphate dehydrogenase (G3PDH) were 5'-TCCACCACCCTGTTGCTGTA-3' and 5'-ACCACAGTCCATGCCATCAC-3' , respectively. PCR products were subjected to 1% agarose gel electrophoresis.
3
Quantifying HHLA2 Expression by qRT-PCR
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Total RNA was extracted from collected tissues and cultured cells by Trizol (Life Technologies, USA) and synthesized cDNA with a reverse transcription-polymerase chain reaction kit (TaKaRa, Japan) according to the manufacturers’ instructions. The quantitation of HHLA2 (primer forward 5′-GGAACACTTCATTTTCCCCAATTC-3′, reverse 5′-TCTCCTACATGCTCTCCTTCCT-3′) was performed with the help of the SYBR Green Master Mix kit and a GAPDH (primer forward 5′-CTGTCGGSCCTGGCCAAG-3′, reverse 5′-CACCCTCGCCAAAGGTGA-3′) as the internal reference. The 2−ΔΔCT method was used to calculate the expression level of HHLA2.
4
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) as per the manufacturer's instructions. A total of 1 mg total RNA was synthesized into cDNA in a 20 ml reaction buffer using a reverse transcription polymerase chain reaction kit (Takara, Kyoto, Japan). The sequences of the primers are shown in Supplemental Table S4. Thermal cycling conditions included 40 cycles at 95 C for 30 seconds, 95 C for 5 seconds, and 60 C for 40 seconds.
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