QuantiTect Reverse Transcription Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Prepare the reverse-transcription master mix on ice and later incubate for 60 min at 37°C. Incubate for 5 min at 95°C to inactivate Quantiscript Reverse Transcriptase and later use it for downstream experiments

Protocol tips
Prepare the reverse-transcription master mix on ice and later incubate for 60 min at 37°C. Incubate for 5 min at 95°C to inactivate Quantiscript Reverse Transcriptase and later use it for downstream experiments

Publication protocol

RT-qPCR was performed to examine the gene expression levels of CFHR3, Ki67, survivin, caspase-3, Bcl-2-associated X (Bax), B cell lymphoma 2 (Bcl-2), PI3K, Akt and mTOR. Total RNA was extracted from tissues samples or cultured cells using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.), according to manufacturer's instructions and reversed transcribed to cDNA using the QuantiTect Reverse Transcription kit (Qiagen GmbH, Hilden, Germany). The RT conditions were as follows: 37°C for 60 min and 95°C for 5 min. qPCR was carried out using the SYBR qPCR Mix (Toyobo Life Science, Osaka, Japan), with GAPDH as the internal reference. The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 10 sec and 72°C for 10 sec. The primer sequences are listed in Table I. The relative mRNA expression levels were calculated using the 2−ΔΔCq method (28).

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Manufacturer protocol

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