Mirna extraction kit

Invitrogen TRIzol^® reagent (Thermo Fisher Scientific, Inc.) was used to isolate total RNA from cells or tissues, according to the manufacturer's protocol. The PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd.) was used for conversion of RNA into cDNA; the miRNA extraction kit of Guangzhou RiboBio Co., Ltd. was used to achieve the same objective with respect to miRNA. Quantification of transcripts was accomplished by performing RT-qPCR using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.)., and the ABI StepOne Plus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to perform qPCR using the SYBR-Green PCR Kit (Takara Biotechnology Co., Ltd.). The qPCR protocol consisted of the following steps: a) initial denaturation for 5 min at 95°C; and b) 40 cycles involving denaturation at a temperature of 95°C for 10 sec, followed by annealing and extension at 60°C for 34 sec. For miR-22 expression in RT-qPCR, U6 was used as the reference gene. These experiments were repeated three times. miR-22 and U6 primers were amplified by a Bulgeloop miRNA RT-qPCR kit (Guangzhou RiboBio Co., Ltd.). Detailed information of the primers employed for the qPCR experiments is shown in Table I, whereas the thermocycling conditions used for the qPCR experiments are shown in Table II.

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate total RNA from cells, according to the manufacturer's protocol. The PrimeScript RT Reagent kit (Takara Bio, Inc.) was used to convert RNA into cDNA. The condition of RT was 15 min at 37°C and 5 sec at 85°C. The miRNA Extraction kit (Guangzhou RiboBio Co., Ltd.) was used to convert miRNA into cDNA. Quantification of the transcript expression levels were obtained by performing qPCR using SYBR Premix Ex Taq (Takara Bio, Inc.). The ABI StepOne Plus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for qPCR using the SYBR-Green PCR kit (Takara Bio, Inc.). The qPCR protocol consists of the following steps: i) Initial denaturation for 5 min at 95°C; and ii) 40 cycles involving denaturation at 95°C for 10 sec, followed by annealing and extension at 60°C for 34 sec. Each experiment was repeated three times. The miR-22 primers used were from the Bulgeloop miRNA qRT-PCR kit (Guangzhou RiboBio Co., Ltd.). Detailed information of the primers employed for the qPCR experiments is presented in Table I. The 2^−ΔΔCq analysis method was used as quantification method (29 (link)).