To further verify the reliability and accuracy of the transcriptomic data, we used quantitative real-time PCR (qRT-PCR) for verification. Alkaline phosphatase-like (ALP-like), trypsin-1-like, C-type lectin domain family 4 member G-like, transcript variant X1 (Clec4g-like, transcript variant X1), crustacyanin-C1 subunit-like, phosphoenolpyruvate carboxykinase, cytosolic [GTP]-like, transcript variant X1 (PCK, cytosolic [GTP]-like, transcript variant X1), protein argonaute-3-like (AGO-3-like) and chymotrypsin BI-like(CHT BI-like) were selected for qRT-PCR analysis. Table 1
Hiscript q rt supermix for qpcr with gdna wiper
Manufactured by Vazyme
Sourced in China
HiScript Q RT SuperMix for qPCR with gDNA wiper is a reverse transcription and real-time PCR reagent kit. It enables reverse transcription and real-time PCR amplification in a single step.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using hiscript q rt supermix for qpcr with gdna wiper
1
Transcriptomic Data Verification via qRT-PCR
2
Quantitative Analysis of CircRNA Expression
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Total RNA was isolated from the cell lines with TRIzol reagent (Invitrogen, CA, USA) following the manufacturer’s instructions. The concentration and purity of the total RNA samples were assessed using a NanoDrop spectrophotometer (Thermo, Wilmington, DE, USA). Total RNA was reverse transcribed using HiScript Q RT SuperMix for qPCR with gDNA wiper (Vazyme Biotech, Nanjing, China), and qPCR assays were performed in triplicate using the AceQ qPCR SYBR Green Master Mix kit (Vazyme Biotech, Nanjing, China) on a 7500 real-time PCR system (ABI). The divergent primers used for detecting circRNAs were synthesized from Shanghai Generay Biotech (Shanghai, China), and β-actin was used as an internal control. The following primer pairs were used for qPCR: β-actin forward, 5′-AGAAAATCTGGCACCACACC-3′ and reverse, 5′-CAGAGGCGTACAGGGATAGC-3′; hsa_circ_0001013 forward, 5′-GTCAAAGGAAGCAAAAGAAAGTCT-3′ and reverse, 5′-GATCGCACCTCTACACTCCA-3′; hsa_circ_0007376 forward, 5′-ATCGACTCCATGGCCAACTC-3′ and reverse, 5′-AAGCCCCGGAGAACAGC-3′; hsa_circ_0043947 forward, 5′-CAATTGTGGTTGTGCAGCC-3′ and reverse, 5′-ACACAAACTCAGCATCATGGA-3′. The expression of circRNAs was normalized to that of the internal control β-actin by using the 2-ΔΔC method [19 (link)].
3
Quantifying CircRNA Expression in Cell Lines
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Total RNA was isolated from the cell lines with TRIzol reagent (Invitrogen, CA, USA) following the manufacturer's instruction. The concentration and purity of the total RNA samples were assessed using the NanoDrop spectrophotometer (Thermo, Wilmington, DE, USA). Total RNAs were reversely transcribe using HiScript Q RT SuperMix for qPCR with gDNA wiper (Vazyme Biotech, Nanjing, China), and qPCR assays were performed in triplicate using AceQ qPCR SYBR Green Master Mix kit (Vazyme Biotech, Nanjing, China) on 7500 real time PCR system (ABI). The divergent primers used for detecting circRNAs were synthesized from Shanghai Generay Biotech (Shanghai, China), and β-actin was used as an internal control. The following primer pairs were used for qPCR: β-actin forward, 5'-AGAAAATCTGGCACCACACC-3' and reverse, 5'-CAGAGGCGTACAGGGATAGC-3'; hsa_circ_0001013 forward, 5'-GTCAAAGGAAGCAAAAGAAAGTCT-3' and reverse, 5'-GATCGCACCTCTACACTCCA-3'; hsa_circ_0007376 forward, 5'-ATCGACTCCATGGCCAACTC-3' and reverse, 5'-AAGCCCCGGAGAACAGC-3'; hsa_circ_0043947 forward, 5'-CAATTGTGGTTGTGCAGCC-3' and reverse, 5'-ACACAAACTCAGCATCATGGA-3'. The expression of circRNAs was normalized to that of internal control β-actin by using the 2 -ΔΔC method [19] .
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