Total RNA was isolated using TRIzol (Invitrogen) following the manufacturer's protocol. First‐strand cDNA synthesis from total RNA was carried out using GoScript™ Reverse Transcription Mix with random primers (Promega), followed by quantitative PCR (qPCR) using AriaMx Real‐Time PCR machine (Agilent Technologies). All RT‐qPCRs were repeated at least three times, and the relative abundance of each transcript was normalized to the expression level of ACTIN. Sequence information for all primers used to check both gene expression and circPVT1 expression was presented in Table EV1 .
Goscript reverse transcription mix with random primers
Manufactured by Promega
The GoScript™ Reverse Transcription Mix with random primers is a reagent designed for cDNA synthesis from RNA samples. The mix contains all the necessary components for the reverse transcription reaction, including the GoScript™ Reverse Transcriptase enzyme and random primers.
Automatically generated - may contain errors
2 protocols using goscript reverse transcription mix with random primers
1
Quantitative RNA Expression Analysis
2
RNA Extraction and qPCR Analysis
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RNA was extracted from microdevice via direct lysis. Subsequent RNA extraction was performed per manufacturer's instructions with the ReliaPrep RNA Cell Miniprep System (Promega Z6010) and eluted in nuclease‐free water and stored at −80°C. RNA concentration was quantified by Nanodrop and then reverse transcribed into cDNA using the GoScript Reverse Transcription Mix with Random Primers (Promega A2801) per the manufacturer's protocol. cDNA was diluted 1:5 in nuclease free water and stored at −20°C. qPCRs were performed using primers and FAM/TAMRA probes from IDT (Integrated DNA Technologies, USA) and PrecisionPLUS 2X qPCR MasterMix with Low ROX (PrimerDesign, UK) as per manufacturer's instructions. Relative gene expression was normalized to two housekeeping genes (β‐actin and TBP) and then benchmarked to control devices.
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