Sparkeasy cell rna kit

Total RNA was extracted from trypsinized cell suspensions using the SPARKeasy
Cell RNA Kit (SparkJade, China) according to the manufacturer’s instructions.
After quantitation using a NanoDrop spectrophotometer (Thermo scientific), 1 μg
of total RNA was used as template for reverse transcription according to the
PrimeScript RT kit (Takara, RR037A, Japan). The cDNA was then diluted and used
as a template for quantitative PCR (qPCR) reactions using the 2 × SYBR Green
qPCR Mix (Spark Jade, AH0104-B, China). Reactions were performed on the Step
OnePlus real-time PCR system (Applied Biosystems, USA) and the relative
expression levels of the target gene mRNA determined against the GAPDH
housekeeping gene control using the 2–ΔΔCt method. The primer sequences are as follows:

GAPDH F:GGAGCGAGATCCCTCCAAAAT;
R:GGCTGTTGTCATACTTCTCATGG

Col1 F: GTTGCTGCTTGCAGTAACCTT; R:
AGGGCCAAGTCCAACTCCTT;

FN F: CGGTGGCTGTCAGTCAAAG; R: AAACCTCGGCTTCCTCCATAA;

αSMA F: AAAAGACAGCTACGTGGGTGA; R:
GCCATGTTCTATCGGGTACTTC;

NOX1 F: TTGTTTGGTTAGGGCTGAATGT; R:
GCCAATGTTGACCCAAGGATTTT;

NOX2 F: GGGCTGTTCAATGCTTGTGGCT; R:
ACATCTTTCTCCTCATCATGGTGC;

NOX3 F: ACCGTGGAGGAGGCAATTAGA; R:
TGGTTGCATTAACAGCTATCCC;

NOX4 F: CAGATGTTGGGGCTAGGATTG; R:
GAGTGTTCGGCACATGGGTA;

NOX5 F: ACTCAGCAGTTTAAGACCATTGC; R:
GGACTCTTTCACATGCAGAGC;

Total RNA was extracted using a SPARK easy Cell RNA Kit (AC0205, Sparkjade, China,) and cDNA was synthesized using a SPARK script II RT Plus Kit (AG0304, Sparkjade, China) according to the manufacturer’s protocol. Subsequently, the quality of the extracted RNA was detected. The primer sequences were as follows: SPHK1 (forward) 5'-CTGTCACCCATGAACCTGCTGTC-3' and (reverse) 5'-ACGCCGATACTTCTCACTCTCTAGG-3', P4HA2 (forward) 5'-CCAGGAACCAAGTACCAGGCAATG-3' and (reverse) 5'-CTGCTCCATCCACAACACCGTATG-3', GAD1 (forward) 5'-TCCTCCTGGAAGTGGTGGACATAC-3' and (reverse) 5'-AGCAACTGGTGTGGGTGATGAAAG-3', AKR1B1 (forward) 5'-TGACACCAGAACGCATTGCTGAG-3' and (reverse) 5'-AGACCCTCCAGTTCCTGTTGTAGC-3' and PTGES (forward) 5'-TCCTGGGCTTCGTCTACTCCTTTC-3', and (reverse) 5'-TGTAGGTCACGGAGCGGATGG-3' and GAPDH (forward) 5'-CCTTCCGTGTCCCCACT-3', and (reverse) 5'-GCCTGCTTCACCACCTTC-3'. Real-time PCR was performed using a SYBR qPCR SuperMix Plus (R311-02, Vazyme, China). The thermocycling conditions were as follows: Initial denaturation at 95 ℃ for 2 min, followed by 40 amplification cycles at 95 ℃ for 15 sec and 60 ℃ for 10 sec. GAPDH was used as the endogenous control for normalization, and the expression was analyzed using the 2‑^ΔΔCT method. The data were extracted from three independent biological experiments with three technical replicates in each experiment.

The HUVECs were seeded in 6-well plates at 2 × 10⁴ cells/well with CON, PTi, AMC/PTi, and AMCI/PTi scaffolds at 37 °C and 5 % CO₂. After culturing for 2 days, an RT-qPCR assay was performed to detect the expression of VEGF, BFGF, and HIIF-1 in HUVECs. The total RNA was extracted using a SPARK Easy Cell RNA Kit (Sparkjade, Shandong, China), and genomic DNA clearance and reverse transcription were performed using a StarScript II RT Kit (Genestar, Beijing, China). Then, the amplification process was performed using a StarScript II One-step RT-qPCR Kit (Genestar, Beijing, China). The relative gene expressions were calculated via the 2^−ΔΔCt method by normalizing with a housekeeping gene. In addition, the BMSCs were seeded at 6 × 10³ cells/well in 24-well plates and cultured in an incubator at 37 °C and 5 % CO₂. After the BMSCs adhered to the plate, 50 mM ascorbic acid, 10 mM β-glycerophosphoric acid, and 100 nM dexamethasone were added to each group. The expression levels of Runx-2, osteocalcin (OCN), and ALP in BMSCs were detected using the above methods after culturing for 14 days. The sequence of primer fragments used in the experiment is shown in Table A1.

Total RNA was isolated and purified from 40 pairs of tissues and TPC-1 and BCPAP cells using TRIzol (Yeasen, Shanghai, China) or the SPARKeasy Cell RNA Kit (Shandong Sparkjade Biotechnology Co., Ltd. China), according to the manufacturer's instructions. qRT-PCR analysis of samples was performed using Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) and Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen, Shanghai, China), following the manufacturer's instructions. β-Actin was used as the control gene. The target genes and primers were designed as follows: eEF1A2 forward: 5′-GTCAAGGAAGTCAGCGCCTA-3′ and reverse: 5′-CTTGAACACGGCATGTTGG-3′; LDHA forward 5′-CTTGGCAGATGAACTTGCT-3′ and reverse 5′-ATGACCAGCTTGGAGTTTG-3′; GLUT1 forward 5′-GTCGTCGGCATCCTCAT-3′ and reverse 5′-TTCTCCTCGTTGCGGTT-3′; HK2 forward 5′-CTGAAGGAAGCGATCCAC-3′ and reverse 5′-CCAACAATGAGGCCAACT-3′; PKM2 forward 5′-CATCCATTAGGCCAGCAAC-3′ and reverse 5′-GCATTCCTCCTTCTTCCCT-3′; β-actin forward 5′-CCTGGCACCCAGCACAAT-3′ and reverse 5′-GGGCCGGACTCGTCATAC-3′.