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The following protocol is for total RNA isolation from cells using the Total RNA Purification Kit (Catalog #79040). For complete instructions, refer to the Technical Manual (Document #10000005434).

Directions

A. Preparation of Cell Lysate

Prepare cell lysate from adherent cells or cells in suspension, as indicated below.

From Adherent Cells

1. Aspirate cell culture medium. Add ice-cold sterile D-PBS as indicated in Table 1. Aspirate D-PBS.



Table 1. Recommended Volumes of PBS, RNA Lysis Buffer + TG, and 100% Isopropanol for Various Cultureware

Cultureware

Volume of D-PBS

Volume of RNA Lysis Buffer + TG

Volume of 100% Isopropanol

T-25 cm² flask

5 mL

500 µL

170 µL

6-well plate

2 mL

250 µL

85 µL

24-well plate

500 µL

100 µL

35 µL

48-well plate

250 µL

100 µL

35 µL

96-well plate

100 µL

100 µL

35 µL


2. Add RNA Lysis Buffer + TG as indicated in Table 1. Gently rock the plate or flask to completely cover the adherent cells with buffer. Pipette the lysate up and down over the cultureware surface 7 – 10 times.
3. Collect the lysate and transfer to a new microcentrifuge tube.
4. Add 100% isopropanol as indicated in Table 1. Mix by vortexing for 5 seconds.
5. Proceed to RNA isolation.

From Cells in Suspension

1. In a sterile centrifuge tube, centrifuge cell suspension at 300 x g for 5 minutes. Remove and discard supernatant.
2. Add ice-cold, sterile D-PBS to wash cells. Centrifuge at 300 x g for 5 minutes. Remove as much supernatant as possible and discard.
3. Add RNA Lysis Buffer + TG as indicated in Table 2. Mix well by pipetting up and down 7 - 10 times, or by vortexing. For > 2 x 10⁶ cells, pass the lysate through a 20-gauge needle 4 - 5 times to shear the genomic DNA.
4. Add 100% isopropanol as indicated in Table 2. Mix by vortexing for 5 seconds.



Table 2. Recommended Volumes of RNA Lysis Buffer + TG and Isopropanol per Cell Input Range

Cell Input Range

Volume of RNA Lysis Buffer + TG

Volume of 100% Isopropanol

1 x 10² to 5 x 10⁵

100 µL

35 µL

> 5 x 10⁵ to 2 x 10⁶

250 µL

85 µL

> 2 x 10⁶ to 5 x 10⁶

500 µL

170 µL


5. Proceed to RNA isolation.

B. RNA Isolation

1. Insert minicolumn into Collection Tube.
2. Transfer the lysate to the minicolumn assembly.
3. Centrifuge at 12,000 - 14,000 x g for 30 seconds. Discard the liquid in the Collection Tube and reinsert minicolumn into Collection Tube.
4. Add 500 μL RNA Wash Buffer to the minicolumn. Centrifuge at 12,000 - 14,000 x g for 30 seconds. Discard the liquid in the Collection Tube and reinsert minicolumn into Collection Tube.
5. Prepare DNase I Incubation Mix by combining the reagents as indicated in Table 3, per sample, in the order listed.



Table 3. Preparation of DNase I Incubation Mix

Component

Volume per Sample

DNA Digestion Buffer

24 µL

MnCl₂

3 µL

DNase I Solution

3 µL


6. Mix by gently pipetting up and down; do not vortex. Store on ice.
7. Add 30 μL of fresh DNase I Incubation Mix directly to the minicolumn membrane. Incubate at room temperature (15 - 25°C) for 15 minutes.
8. Add 200 μL of Column Wash Buffer (with ethanol added) to the minicolumn. Centrifuge at 12,000 - 14,000 x g for 15 seconds.
9. Add 500 μL of RNA Wash Buffer (with ethanol added) to the minicolumn. Centrifuge at 12,000 - 14,000 x g for 30 seconds. Remove the minicolumn and transfer to a new Collection Tube. Discard the Collection Tube containing wash buffer.
10. Add 300 μL of RNA Wash Buffer to the minicolumn. Centrifuge at high speed for 2 minutes.
11. Transfer minicolumn to an Elution Tube. Add nuclease-free water to the minicolumn membrane as indicated in Table 4. Incubate at room temperature for 1 - 2 minutes. Centrifuge at 12,000 - 14,000 x g for 1 minute.



Table 4. Recommended Volume of Nuclease-Free Water per Input Cell Range

Cell Input Range

Volume of Nuclease-Free Water

1 x 10² to 5 x 10⁵

15 µL

> 5 x 10⁵ to 2 x 10⁶

30 µL

> 2 x 10⁶ to 5 x 10⁵

50 µL


12. Remove and discard minicolumn. Store purified RNA at -80ºC.

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