Abi 7300 rt pcr machine

We used the KAPA SYBR FAST qPCR-Kit (KAPA, cat. No. KK4618), TB Green Premix Ex Taq™ II (Takara, cat. No. RR820B) and followed the manufacturer's standard protocol to perform RT PCR. The RT-PCR reaction was analyzed in the ABI-7300 RT-PCR machine (Applied Biosystems, CA, USA). The level gene transcripts were normalized with the ACT1 transcript level, and a relative fold change in gene expression was calculated as the 2 -ΔΔCT method (101).

For quantitative real-time polymerase chain reaction (qRT-PCR), the total RNAs, including the miRNAs, were harvested from the sorted cell using TRIzol Reagents (Invitrogen, Carlsbad, CA) according to the manufacturer's specifications. The total RNA was quantified with a spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Approximately 2 µg of the total RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) according to the manufacturer's instructions. Afterward, qRT-PCR was performed to validate the miRNA expression level. The expression levels of mature miRNAs were then amplified using SYBR Green qRT-PCR on an ABI7300 RT-PCR machine (Applied Biosystems, Foster City, CA, USA). For the miRNA quantification, a Bulge-Loop ™ miRNA qRT-PCR primer set (one RT primer and a pair of qPCR primers for each set) specific for miR-149-5p, miR-6865-3p, miR-4725-5p, miR-1825, let-7b-3p, miR-8089, miR-572, miR-6734-5p, and miR-3648, which were designed by RiboBio Co., Ltd. U6 was used as the housekeeping gene to measure the levels of the representative miRNAs. The relative levels of gene expression were determined using the ΔΔCt method. All the reactions were repeated three times for each sample. In addition, three individual experiments were repeated.