RNeasy Plus Micro Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- "When processing <2 µg tissue, carrier RNA may be added to the lysate. Foaming can be reduced by adding Reagent DX at a final concentration of 0.5% (v/v) before disruption and homogenization. - Add 4 volumes of ethanol (96–100%) to Buffer RPE for a working solution. before homogenization

Upstream tips
- "When processing <2 µg tissue, carrier RNA may be added to the lysate. Foaming can be reduced by adding Reagent DX at a final concentration of 0.5% (v/v) before disruption and homogenization. - Add 4 volumes of ethanol (96–100%) to Buffer RPE for a working solution. before homogenization

Publication protocol

Total RNA was isolated from each type of tissue using an RNeasy Plus Mini Kit (Qiagen, Tokyo, Japan) and was stored at −80°C until used.

Real-time quantitative RT-PCR (qRT-PCR) was performed with a Thermal Cycler Dice Real Time System TP800 (Takara) using One Step SYBR® PrimeScrip PLUS RT-PCR kit (Takara). The primer pairs for qRT-PCR were 5′-TCCAATTTAGGAGAGCCAAGC-3′ and 5′-GCCGACATCAGTCCACATAG-3′ for mouse Prnp mRNA and 5′-AGCCAAGCACATACACCAAA-3′ and 5′-GGGTTTAGACCGTCGTGAGA-3′ for 28S rRNA, respectively. The PCR amplification was carried out in a 25 µl reaction mixture containing 5 ng (for 28S rRNA) or 50 ng (for Prnp) of total RNA, 0.4 µM of each primer, 12.5 µl of 2 ×One Step RT-PCR Buffer 4, 1.5 µl of TaKaRa Ex Taq HS Mix and 0.5 µl of PrimeScript PLUS RTase Mix. The PCR conditions consisted of an initial reverse transcription step for 5 min at 42°C and a polymerase activation step for 10 sec at 95°C, followed by 45 cycles of 95°C for 5 sec and 65°C for 30 sec. Both dissociation curve analysis and agarose gel electrophoresis were used to check the specificity of the qRT-PCR.



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Manufacturer protocol

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