Sybr green pcr mix

Total RNA was extracted from cells using TRIzol regent (#335904; Invitrogen Corp., Carlsbad, CA, USA), and RNA was reverse transcribed to complementary DNA (cDNA) using a GoScript RT Reagent Kit (#A5001; Promega Corp., Fitchburg, WI, USA). We performed RT‐qPCR using a QuantStudio 6 Flex RT‐qPCR System (Applied Biosystems, Foster City, CA, USA) with SYBR Green PCR Mix (#B21703; Bimake, Houston, TX, USA). All RT‐qPCR primers are listed in Table S1.

Total RNA was extracted using an RNA extraction kit (TIANGEN, Beijing, China) and reverse-transcribed into cDNA using an iScript cDNA synthesis kit (TaKaRa, Tokyo, Japan). The primers used are listed in Table 1. The real-time polymerase chain reaction was performed using SYBR Green PCR mix (Bimake, San Francisco, CA, USA) in a real-time PCR apparatus (Bio-Rad, Hercules, CA, USA). GAPDH was used as an internal control. The accession numbers are as follows: p53 NM_030989.3, p21 U24174.1, p16 L81167.1, and GAPDH NM_017008.4.

Total RNA was meticulously extracted from cultured cells using the AxyPrep Multisource RNA Miniprep Kit (Axygen, Corning, New York, USA). Briefly, after cell lysis, we performed centrifugation and washing steps using various reagents from the kit. Finally, we added TE buffer to dissolve the RNA, followed by centrifugation to obtain the RNA liquid sample. The synthesis of complementary DNA (cDNA) was conducted via reverse transcription using TaKaRa reverse transcription reagents (TaKaRa, Shiga, Japan). Subsequently, RT-qPCR assays were carried out utilizing the QuantStudio 6 Flex RT-qPCR System (Applied Biosystems, CA, USA) in conjunction with SYBR Green PCR Mix (Bimake, TX, USA). The detailed primer sequences are shown in Additional file 1: Table S1. In this experiment, NP cells were subjected to different treatments as required by the experimental demands, as detailed in the Results section.