Kaneka Easy RNA Extraction Kit (for RT-PCR)

Product#: KN-T110004
$480.00
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Kaneka Easy RNA Extraction Kit (for RT-PCR)



Cat No: KN-T110004
 
Size: 1 kit
Storage: -20°C

Description

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.

For Cultured cells

Prepare a cell suspension of 103-106 cells/ml and put it in a centrifuge tube.
Centrifuge the tube at 1000 rpm for 3 minutes. Gently remove the supernatant of the suspension without removing the cell pellet.
Add 100 μl of PBS (-) and resuspend the cells by pipetting. Centrifuge the tube at 1000 rpm for 3 minutes. Remove the supernatant and collect the cells.
Add 20 μl of Reagent A to the tube containing the cell, and mix well by pipetting to suspend. Transfer the suspension to a new PCR tube.
Incubate the PCR tube containing the suspension 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

For Blood

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.

Examples

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).
 
KN-T110004_fig1.png

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).
KN-T110004_fig2.png 
Fig 3: Result of PCR/RT-PCR with extracted RNA from mouse blood

DNA/RNA extraction kits
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