Revertaid first strand complementary dna cdna synthesis kit

Total cellular RNA was extracted from ALDH^high and ALDH^low cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Total RNA (500 ng) was reverse transcribed using the RevertAid™ First-Strand Complementary DNA (cDNA) Synthesis Kit (Thermo Scientific). Following cDNA synthesis, RT-PCR was performed in triplicate for each sample using FAST SYBR™ Green detection chemistry (Applied Biosystems) on Step One instrument. Human SOX2, NANOG, and GAPDH were amplified using gene-specific primers (GAPDH: forward primer 5′-ACATCGCTCAGACACCATG-3′, reverse primer 5′TGTAGTTGAGGTCAATGAAGGG-3′; SOX2: forward primer 5′-GGAAACTTTTGTCGGAGACG-3′, reverse primer 5′-GCAGCGTGTACTTATCCTTC-3′; NANOG: forward primer 5′AGAAATACCTCAGCCTCCAG-3′, reverse primer 5′-CGTCACACCATTGCTATTCTT-3′). The cycling parameters consisted of denaturation at 95°C for 10 min; and 40 cycles of 15 s at 94°C, 30 s at 60°C, and 1 min at 72°C; followed by a continuous melting curve.

The rat basophilic leukemia cell line, RBL-2H3 cells, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and seeded in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), and NP, NPA, or NPR extracts (10 μg/mL) for 30 min and then stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (PI) for 16 h. RNA was extracted using an RNeasy mini kit (Qiagen, Hilden, Germany) and reverse-transcribed using a RevertAid first-strand complementary DNA (cDNA) synthesis kit (Thermo Fisher Scientific, Bremen, Germany). Real-time PCR was conducted using a QuantaStudio 6 pro (Thermo Fisher Scientific) using SYBR1 Green (Power SYBR Green PCR Master Mix, Applied Biosystems, Foster City, CA, USA). The primer sets used were IL-4 (accession no. AY496861), forward 5′-ACC TTG CTG TCA CCC TGT TC-3′ and reverse 5′-TTG TGCA GCG TGG ACT CAT TC-3′; β-actin (accession no. EF156276.1), forward 5′-CAC GGC ATT GTC ACC AAC TG-3′ and reverse 5′-AAC ACA GCC TGG ATG GCT AC-3′. Transcript levels were normalized versus glyceraldehydes-3-phosphate dehydrogenase (GAPDH).

RNA was extracted from 5 × 10⁶ FMCm cells using an RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Then, 500 ng of total RNA was reverse-transcribed using the RevertAid First Strand complementary DNA (cDNA) Synthesis kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Primers were designed with PRIMER-BLAST software, National Center for Biotechnology Information (NCBI) on an exon–exon junction (TFRC F5 forward primer: GGTGCCAGTGTCACAAAACC; TFRC F3 reverse primer: ATGCCACATAGCCCTCTGGA). RT-PCR was carried out using a Phire™ Hot Start II DNA Polymerase (Thermo Fisher Scientific) according to the manufacturer’s instructions (Mj research PTC-200 Thermal cycler). The expected PCR product (526 bp) was visualized by 2% agarose gel electrophoresis and was then treated with Exosap-IT PCR product cleanup (Applied Biosystem, Foster City, CA, USA) to allow purification for sequencing. Sanger sequencing was performed by BMR Genomics (University of Padua, Italy).

Chondrocytes were seeded onto 6-well plates at the density of 2.0 × 10⁵ cells/well. After the indicated treatment, RNA samples were isolated with Trizol reagent (Thermo Fisher Scientific, Foster City, CA, USA). The reverse transcription was conducted using a commercial RevertAid First-Strand complementary DNA (cDNA) Synthesis kit (Thermo Fisher Scientific) and stem-loop reverse-transcription universal primer (Ribobio, Guangzhou, China). cDNA was amplified using RT-qPCR mix (Thermo Fisher Scientific) and specific primers (Table 1). The fold changes were assessed by the 2 (-Delta Delta C(T)) method [21 (link)] with the references of glyceraldehyde-phosphate dehydrogenase (GAPDH; for circRNAs and mRNAs) and U6 (for miRNAs).Table 1

Primer sequences in RT-qPCR

Gene	Forward primer (5′–3′)	Reverse primer (5′–3′)
circ_0043947	ATCATTCACCCTTGGCACAG	CATTTTCCTCCCGCAATTC
BRCA1	TTCACCCTCTGCTCTGGGTA	TGGTCACACTTTGTGGAGACA
miR-671-5p	GCCGAGAGGAAGCCCTGGAGGGG	CTCAACTGGTGTCGTGGA
RTN3	CATCTCCTCGTCGTCCTTCG	AATCAGATCGTGCACCGCA
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT
GAPDH	GAAAGCCTGCCGGTGACTAA	TTCCCGTTCTCAGCCTTGAC

Extraction of total RNA from tissues and cells was via TRIzol reagent (Thermo Fisher Scientific, Inc.). Reverse transcription of RNA was via RevertAid First Strand complementary DNA (cDNA) Synthesis Kit (K1622, Thermo Scientific). RT-qPCR was via SYBR Green PCR Master Mix (Applied Biosystems, USA) and BioRad CFX-96 real-time PCR system (BioRad, USA). Primers were manifested in Table 2. Normalization of miRNA and mRNA was to U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), separately. Calculation of the relative RNA was via 2^−ΔΔCT method [26 (link)].Table 2.

RT-qPCR primer sequence

Genes		Forward (5’-3’)	Reverse (5’-3’)
BRD4	Mice	GGACGAGGGAGGAAAGAAACA	AGGAAAGGGGTGAGTTGTGG
	Human	GTGGTGCACATCATCCAGTC	CCGACTCTGAGGACGAGAAG
GAPDH	Mice	TGGCCTTCCGTGTTCCTAC	GAGTTGCTGTTGAAGTC
	Human	TGTGGGCATCAATGGATTTGG	ACACCATGTATTCCGGGTCAAT
MiR-103a-3p	Mice	AGCAGCATTGTACAGGGCTATGAA	TGGTGTCGTGGAGTCG
	Human	AGCAGCATTGTACAGGGCTA TGAA	TGGTGTCGTGGAGTCG
U6	Mice	TCGCACAGACTTGTGGGAGAA	CGCACATTAAGCCTCTATAGTTACTAGG
	Human	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT

Total RNAs were extracted from appropriate maize tissues using the TRIzol reagent according to the manufacturer’s specifications (TakaRa, China). The RNA samples were reverse-transcribed using the RevertAid First Strand complementary DNA (cDNA) Synthesis Kit (Thermo Scientific, USA). Gene-specific primers for maize NAC genes were designed with the Roche LCPDS2 software. The primer sequences used in the RT-qPCR are listed in Supplementary Table 1. RT-qPCR was performed using a LightCycler^® 480 II Real-time PCR Instrument (Roche, Swiss) with SYBR Premix Ex TaqTM II (TakaRa, China). Each sample was run in triplicate for analysis. The maize GAPDH (NM_001111943) gene was used for RT-qPCR as an internal control for normalization, and the 2^-ΔΔCt method was used to calculate the relative expressions of NAC genes under different stress treatments.