RNA extract applicable for RT-PCR can be obtained by simply adding the reagents to the sample (blood, cultured cells, etc.) and heating it. The operation requires about 30 minutes and since no organic solvent or special equipment is necessary, it shortens processing time and make extraction easier than the spin column method.

Prepare a cell suspension of 10³-10⁶ cells/ml and put it in a centrifuge tube.

Add 20 μl of Reagent A to a PCR tube.
Add 0.5-2.0 μl of fresh blood mixed with anticoagulant agent*3 such as heparin to the tube and mix well by pipetting.
Incubate the tube at 75℃ for 5 minutes in a heat block or other heating equipment.
After cooling the tube to room temperature, add 1 μl of Dnase I and mix by pipetting. Incubate it at 42℃ for 10 minutes, and then at 75℃ for 5 minutes.
Use 1-2 μl of the incubated suspension*1 as a template RNA for reverse transcription (for a 25 μl reaction)*2.

1*  You may find some sediments in the incubated suspension depending on the species and condition of the sample. Though it is not strictly required, you can remove such sediments by centrifuging the tube at 5000 rpm for 5 minutes at 4℃ and using the supernatant.

*2 We recommend the immediate use of the obtained suspension for RT-PCR. Keep the suspension on ice while using it, or store it at lower than -20℃.

*3 We have confirmed that RNA can be extracted from blood mixed with either heparin or EDTA.

1- Results of RNA extraction from cultured cells and RT-PCR detection

Extraction of RNA respectively from adherent cells (HEK293T) and floating cells (Jurkat), both of which had been prepared to have 10⁵ cells, using this product. We then used the extract as a template RNA and performed RT-PCR targeting ACTB mRNA using T3000 Thermocycler (Biometra) with PrimeScript® One Step RT-PCR kit Ver. 2 (Takara Bio). We subjected the obtained RT-PCR product to electrophoresis and detected amplified fragments specific to the template RNA (Fig.1, 2). We also used the same extract as a template and performed PCR using T3000 Thermocycler (Biometra) with TaKaRa Ex Taq® (Takara Bio) in order to confirm that the extract did not contain any genomic DNA from the sample. We subjected the obtained PCR product to electrophoresis and confirmed that no amplified fragment was detected (Fig. 1, 2).
 

2- Results of RNA extraction from mouse blood and RT-PCR detection

Extraction of RNA from mouse blood anticoagulated with heparin using this product. We then used the extract as a template RNA and performed RT-PCR targeting H3f3a mRNA using T3000 Thermocycler (Biometra) with PrimeScript® One Step RT-PCR kit Ver. 2 (Takara Bio). We subjected the obtained RT-PCR product to electrophoresis and detected amplified fragments specific to the template RNA (Fig. 3). We also used the same extract as a template and performed PCR using T3000 Thermocycler (Biometra) with TaKaRa Ex Taq® (Takara Bio) in order to confirm that the extract did not contain any genomic DNA from the sample. We subjected the obtained PCR product to electrophoresis and confirmed that no amplified fragment was detected (Fig. 3).