Takara reverse transcription reagents

Total RNA was isolated from P. cyrtonema rhizome (one-year, two-year, three-year, four-year) using the E.Z.N.A. Total RNA Kit I (Omega, USA) and reverse-transcribed to cDNA with TaKaRa reverse transcription reagents (TaKaRa Bio, Dalian, China). The elongation factor 1-ɑ (EF1ɑ, TRINITY_DN27092_c0_g5_i1_1) genes were selected as endogenous references for normalization according to its expression level and stability in transcriptome data. Specific primers were designed by primer 3.0 (Additional file 11: Table S5). Real-time PCR was performed by QuantiNova Syb^r Green PCR kit (Qiagen). The results of the target gene relative to the reference gene were calculated by the 2^-ΔΔCt method [36 (link)]. Data are presented as the mean ± standard deviation (SD) of three reactions performed in different 96-well plates. The data were analyzed using CFX Manager™ v3.0.

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.

The total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) from the DLBCL cells that had been treated with indirubin and As₂S₂, alone or in combination, and from the untreated cells. The reverse transcription reaction step was then performed using Takara reverse transcription reagents (Takara, Dalian, China). The amplification reactions were performed using SYBR Premix Ex Taq (Takara) on a Roche LightCycler 480 Real-Time PCR System (Roche Diagnostics, Basel, Switzerland). Specific primers for the qPCR were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The primer sequences are listed in Table I. In order to control the variability in expression levels, the data were normalized to the geometric mean of the housekeeping gene, β-actin. For the data analysis, the 2^−ΔΔCt method was used. The qPCR for each gene of each cDNA sample was performed in triplicate.

Total RNA was extracted from tissues or cultured cells using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), as previously described [24 (link)]. cDNA was reverse transcribed using TaKaRa reverse transcription reagents (TaKaRa Bio Inc., Shiga, Japan) and PCR was performed using Real-Time PCR Mix (TaKaRa) on a light cycler (Roche, Basel, Switzerland) with the following primers: ColX (forward primer 5′-AAA GCT TAC CCA GCA GTA GG-3′ and reverse primer 5′-ACG TAC TCA GAG GAG TAG AG-3′), MMP13 (forward primer 5′-CTT CTT GTT GAG CTG GA CTC-3′ and reverse primer 5′-CTG TGG AGG TCA CTG TAG ACT-3′), Runx2 (forward primer 5′-TCC CCG GGA ACC AAG AAG GCA-3′ and reverse primer 5′-AGG GAG GGC CGT GGG TTC TG-3′), and GAPDH (forward primer 5′-AGG TCG GTG TGA ACG GAT TTG-3′ and reverse primer 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′). The glyceraldehyde 3-phosphate dehydrogenase gene was used as an endogenous control to normalize the differences in the amount of total RNA.

Total RNA was extracted from cultured cells using TRIzol® reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was reverse transcribed using TaKaRa reverse transcription reagents (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) and RT-qPCR was performed using the Real-Time PCR Mix (TaKaRa Bio Inc.) on a light cycler (Roche Diagnostics) with the following primers: circFOXO3 (forward primer 5′-GTGGGGAACTTCACTGGTGCTAAG-3′ and reverse primer 5′-GGGTTGATGATCCACCAAGAGCTCTT-3′), FOXO3 (forward primer 5′-AAACGGCTCACTTTGTCCCAGATC-3′ and reverse primer 5′-CCTCGGCTCTTGGTGTACTTGTTG-3′), autophagy-related gene 5 (ATG5; forward primer 5′-CATCCACTGGAAGAATGACAG-3′ and reverse primer 5′-TGATGCAAGAAGATCAAATAG-3′), Beclin1 (forward primer 5′-TTTTCTGGACTGTGTGCAGC-3′ and reverse primer 5′-GCTTTTGTCCACTGCTCCTC-3′), microtubule-associated proteins 1A/1B light chain (LC3; forward primer 5′-GATGTCCGACTTATTCGAGAGC-3′ and reverse primer 5′-TTGAGCTGTAAGCGCCTTCTA-3′), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; forward primer 5′-CGACTTCAACAGCAACTCCCACTCTTCC-3′ and reverse primer 5′-TGGGTGGTCCAGGGTTTCTTACTCCTT-3′). Target gene expression levels were determined using the 2^-ΔΔCq method [31 (link)], with GAPDH as the internal reference control.