Hiscript 3 reverse transcriptase

To clone the gene sequence, R. glutinosa cultivar “Wen 85-5” roots were sampled at the root elongation stage (60 days after seedling emergence). Total RNA from R. glutinosa was isolated using TRIzol (Invitrogen, Carlsbad, USA) as recommended by the manufacturer. A Nanodrop 2000 instrument (Thermo Scientific, Wilmington, DE, USA) was used to measure the RNA concentration. A 1 μg aliquot of total RNA was reverse-transcribed into cDNA using Hiscript III Reverse Transcriptase (Vazyme, Nanjing, China) in a total volume of 20 µL using oligo(dT) primers. A putative RgC3H sequence was obtained from R. glutinosa transcriptome data [4 (link)]. Sequence analysis by using BlastN [43 (link)] indicated that it was highly homologous to C3Hs from other plant species. Specific primers (Table S3) of the RgC3H sequence were designed by using oligo 7.0 software, and the cDNA sequence was amplified by PCR using PrimeSTAR^® HS DNA Polymerase (Takara, Tokyo, Japan). The product was purified with the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit and subcloned into the pMD-18 vector (Takara, Tokyo, Japan), which was then used to transform E. coli. All constructs were verified by sequencing (Sangon, Shanghai, China)

RNA samples from the clinical tissue specimens and cell lines used in this study were extracted with TRIzol® reagent (Life technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. First-strand cDNA was synthesized using the Hiscript III Reverse Transcriptase (Vazyme). Relative RNA levels determined by quantitative Real-Time PCR (qPCR) were measured on a 7900 Real-Time PCR System with the SDS 2.3 software sequence detection system (Applied Biosystems, USA) using the ChamQ SYBR qPCR Master Mix (Vazyme) method. The sequences for the gene-specific primers used are listed in Additional file 1: Table S2. GAPDH was employed as an internal control to quantify of LacRNA and the mRNA levels of other genes. The relative levels of RNA were calculated using the comparative CT (2^−ΔΔCT) method.

Total RNA was extracted using the Total RNA Isolation Kit (Vazyme, R401-01, Nanjing, China) and reverse transcribed into cDNA using HiScript III Reverse Transcriptase (Vazyme #R333, Nanjing, China). RT-qPCR was performed to detect the corresponding gene expression. The relative level of change in target genes was calculated using 2^−ΔΔCT. Please refer to Table S3 for the primers used for RT-qPCR.

Total RNAs were extracted with RNA isolater (Vazyme, R401-01), and reverse transcribed using HiScriptIIIReverseTranscriptase (Vazyme, R323-01). qRT-PCR was performed with GAPDH as the house-keeping gene using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-03). Primers for qRT-PCR of genes were designed as primer F at BFP and primer R at genes. The following primers were used in this study.

Total RNA was isolated using RNA Isolater Total RNA Extraction Reagent (Vazyme, Nanjing, China) and dissolved in RNase-free ddH₂O (Vazyme). Reverse transcription and quantitative real-time PCR (qRT-PCR) procedures were performed using Hiscript III Reverse Transcriptase (Vazyme) and ChamQ Universal SYBR qPCR Master Mix (Vazyme). The primers were synthesized by Sangon (Shanghai, China), and the sequences were as follows: ALDH3B1, 5′-GCC CTG GAA CTA TCC GCT G-3′ (forward), 5′-CGT TCT TGC TAA TCT CCG ATG G-3′ (reverse); NCEH1, 5′-GAA TAC AGG CTA GTT CCA AAG-3′ (forward), 5′-TAC TTC TGT AAG ACT TCT GGC-3′ (reverse); GAPDH, 5′-GGA GCG AGA TCC CTC CAA AAT-3′ (forward), 5′-GGC TGT TGT CAT ACT TCT CAT GG-3′ (reverse). The whole experimental process was carried out as described previously by our group (35 (link)). Gene expression was compared using the semiquantitative 2-^ΔΔCt method.

Reverse transcription of one microgram of total RNA for each sample was performed using HiScript^® III Reverse Transcriptase (Vazyme Biotech Co., Ltd., Nanjing, China). Real-time PCR was conducted on a CFX Connect Real-Time system (BIO-RAD) using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China). The TIP41 (tonoplastic intrinsic protein 41) gene was used as the reference, and each sample was assessed in triplicate of technical replication. All primers used are listed in Table S1.

CLIP-qPCR (cross-linking and immunoprecipitation followed by reverse transcription and quantitative PCR) was used to mapping and quantifying PHB2-lacRNA interactions. MDA-MB-231 cells were incubated with DMED culture medium eith 4-Thiouridine (4-SU) added (1 M stock solution in DMSO, to a final concentration of 100 μ min). Irradiate cells with 150 mJ/cm2 of UVA (365 nm), then scrape cells in 5 ml PBS, transfer to 50 ml conical tube, centrifuge at 2000× g at 4 ℃ for 5 min and aspirate the supernatant. Lysis cells in NP-40 buffer and incubate with RNase T. RNA was extracted and purified. Run the RNA samples in 1.5% formaldehyde agarose gel to verify that RNAs are digested in 100- to 300-nt range. The supernatants were incubated with 5 μg PHB2 antibody for 12 h at 4 °C with gentle rotation. Then 50 μl Dynabeads Protein G beads were added to the mixture and incubated for another 2 h at 4 °C. After centrifugation at 2000×g for 1 min at 4 °C, wash beads three times with NP-40 lysis buffer. Incubate the pellets with 20 units of RNase-free DNase I in 100 μl NP-40 lysis buffer for 15 min at 37 °C. Then extracted and purify RNA. For qRT-PCR, reverse transcription was performed with Hiscript III Reverse Transcriptase (Vazyme) followed by qRT- PCR analysis.