Sybr premix ex taq tli rnaseh plus

After treatment, the medium was removed and the cells were washed with PBS. The total RNA of each group was extracted using TRIzol (Invitrogen, California, USA). Then the RNA was reversely transcribed to cDNA using the PrimeScript™ RT Reagent kit (Takara, Dalian, China) according to the manufacturer’s instruction. The qPCR was performed using a SYBR Premix Ex Taq (Tli RNaseH Plus) in Applied Biosystem 7300 (Applied Biosystems, Foster city, CA, USA). The BCL-2 mRNA expression was analyzed using the 2-ΔΔCq method taking β-Actin as reference. The gene primer sequences were shown in Table 1.Table 1

Primer sequences for genes

Gene	Primer Sequences
BCL-2	F: 5’-AACATCGCCCTGTGGATGAC-3’
	R: 5’-AGAGTCTTCAGAGACAGCCAGGAG-3’
β-Actin	F: 5’-CATTGCCGACAGGATGCAG-3’
	R: 5’-CTCGTCATACTCCTGCTTGCTG-3’

The mRNA isolation and qPCR were carried out using the previously reported procedure [22 (link)]. To isolate total mRNA, wound tissues were kept in the Trizol reagent (Invitrogen, Carlsbad, CA). Then, using reverse transcription, 1 μg RNA was used to create cDNA (Thermo Scientific kit; Burlington, Canada). The cDNA samples were then used in an RT-PCR reaction (10ng of cDNA, 9 μl of qPCR Master Mix and 20 μl of respective primers) using SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (Applied Biosystems). The relative gene expression was estimated using Ct values and 2^−ΔΔCT method. Table 1 lists the genes used in the investigation.

Total RNA from UMSCs, B2M^‐UMSCs and their respective exosome preparations were extracted using the TRIzol reagent (Life Technologies). RNA concentrations were measured using a spectrophotometer (NanoDrop). Equal amounts of RNA (1 μg) were reverse‐transcribed using the Revert Aid First Strand cDNA Synthesis kit (Thermo Scientific). The Bulge‐loop^TM miRNA qRT‐PCR Primer Sets (one RT primer and a pair of qPCR primers for each set) specific for has‐miR‐24, cel‐miR‐39 and U6 were designed by RiboBio. The expression levels of miRNAs were analysed by qRT‐PCR using Takara SYBR Premix Ex Taq (Tli RNaseH Plus) in a StepOnePlus Real‐Time PCR system (Applied Biosystems). The Ct values of cells were averaged and normalized to the U6 RNA, and the Ct values of exosomes were averaged and normalized to the cel‐miRNA‐39.32 All experiments were repeated at least three times. Relative expression was determined by the _ΔΔCt comparative threshold method.