RNA extraction experiments from a limited number of cells

Summary

RNA samples can be stored at -80°C for up to 6 months. Total cellular RNA is extracted using the StratageneRNA Extraction Kit, following the instructions. Alternatively, the PicoPureRNA Extraction Kit can be used, especially if there are fewer cells. This experiment is based on the PCR Laboratory Guide (2nd edition) by Kang Seed and Lijia Qu.

Operation method

RNA extraction experiments from a limited number of cells

Materials and Instruments

LCM cap
Ethanol Glycogen RNA Extraction Kit Ultrapure Water
Pipettes

Move

I. Materials

1. Buffers, reagents and solutions

75% Ethanol (prepared with ultrapure water)

Glycogen, 5 mg/ml (Ambion, Austin, TX)

StratageneRNA extraction kit (200345, StratageneCloningSystems, LaJolla,CA).

Includes denaturing solution, saturated phenol (stored at 4°C), β-mercaptoethanol (stored at 4°C), chloroform: isoamyl alcohol,

2mol/L sodium acetate, and isopropanol. Or use the PicoPureRNA Isolation Kit (KIT0202, Arcturus.

MountainView,CA)

DEPC-treated ultrapure water (BiotecxLaboratories, Houston, TX) (4°C storage)

2. Specialty equipment

Pipettes

1.5 ml tubes without RNAase

3. Cells and tissues

LCM cap from protocol 3

II. Methods

1. Add 200ul of denaturing solution and 1.6ul of β-mercaptoethanol to a 0.5 ml tube.

2. Insert LCM cap into tube and vortex for 5s.

3. Invert tube and incubate for 5 min.

4. Remove the LCM cap. The sample obtained in this step can be stored at -80°C for up to 6 months.

5. Transfer the solution to a 1.5 ml tube.

6. Add 2ul of 2mol/L sodium acetate and mix gently.

7. Add 220ul water-saturated phenol (bottom layer) and mix gently.

8. Add 60ul chloroform:isoamyl alcohol and shake vigorously.

9. Place on ice for 15 min.

10. Centrifuge at 14000r/min 4°C for 30 min.

11. Pipette the upper solution into a new tube.

12. Add 2ul of glycogen (final concentration 10~150ug/ml).

13. Add 200ul cold isopropyl alcohol.

14. -80°C overnight.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/