An RNAiso Plus Purification Kit (TaKaRa Biotechnology Co., Ltd, 9108) was used to extract total RNA from the cardiomyocytes. The RNA concentration was evaluated based on the optical density of the sample at 260 nm, and the RNA integrity was assessed through 2% agarose gel electrophoresis. The RNA was reverse-transcribed into cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa Biotechnology Co., Ltd, RR047A) [71 (link), 72 (link)]. Real-time PCR was performed on a LightCycler machine (Roche) with a commercial SYBR Green reaction reagent (TaKaRa Biotechnology Co., Ltd, RR820A). GAPDH was used as an internal control. The cDNA was denatured for 30 s at 95 °C, followed by 40 cycles of 5 s at 95°C [73 (link)].
Sybr green reaction reagent
Manufactured by Takara Bio
SYBR Green reaction reagent is a fluorescent dye used for the detection and quantification of double-stranded DNA in real-time PCR (polymerase chain reaction) experiments. It binds to the minor groove of DNA and emits a fluorescent signal upon binding, allowing for the monitoring of DNA amplification in real-time.
Automatically generated - may contain errors
2 protocols using sybr green reaction reagent
1
RNA Extraction and Real-time PCR Analysis
2
Quantifying Brain Gene Expression
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Total RNA was extracted from brain tissue using the RNAiso Plus Purification Kit (9108; Takara Biotechnology Co., Ltd.). RNA was evaluated by A260 (OD) measurement and integrity was checked by 2% agarose gel electrophoresis. RNA was reverse transcribed into cDNA using PrimeScript™ RT Reagent Kit for gDNA Eraser Reverse Transcript Reagent Kit (RR047A; Takara Biotechnology Co., Ltd.). qPCR was performed in a LightCycler machine (Roche) with commercial SYBR-Green reaction reagent (RR820A; Takara Biotechnology Co., Ltd.). GAPDH was used as an internal control. The conditions for PCR consisted of 40 cycles of 95°C for 5 sec and 60°C for 20 sec followed by extension at 72°C for 10 min. The primer sequences used for gene amplification (Invitrogen; Thermo Fisher Scientific, Inc.) were as follows: MFSD2A-forward, 5′-CCACATTCACCATCCCTATCT-3′ and reverse, 5′-TTCTTATTCTGTCGCCGCTTC-3′, GAPDH-forward, 5′-ATGCCGCCTGGAAACC-3′ and reverse, 5′-GCATCAAAGTGGAAGAATGG-3′. The amplified products were 199 bp in length; gene expression was quantified via the 2−∆∆Cq method (20 (link)).
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