Ly trích RNA dùng cho phân tích qPCR

RNA EXTRACTION

 SỬ DỤNG ATA BUFFER

1. Grind samples in liquid N

2. Add 600 ul extraction buffer

            5-10 ul mercaptoethanil

            2 ul antifoam

3. Transfer suspension to clean eppendorf

4. Add 84ul KCl 3M and keep on ice 15’

5. Centrifuge 8000rpm for 5’ at 4⁰C

6. Transfer supernatant to new tube

7. Add an equal volume of LiCl₂ 8M (finally get LiCl₂ 4M)

8. Keep at 4⁰C over night

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9. Centrifuge 15000 rpm 20’ at 4⁰C

10. Discard supernatant; add 400 ul DEPC H₂O to pellet

11. Vortex, add 40 ul NaOAC 3M then vortex

12. Add 450 ul phenol:chloroform (1:1) then vortex

13. Centrifuge at 15000 rpm for 5’ at room temp.

14. Transfer the upper aqueous phase into new tube

15. Add an equal volume of chloroform

16. Mix and centrifuge at 15000 rpm for 5’ at 4⁰C

17. Transfer the upper aqueous phase into new tube

18. Add 2.5 volume of cold ethanol absolute

19. Add 10 ul NaOAC

20. Keep -20⁰C for 30’

21. Centrifuge 15000 rpm 10’ at 4⁰C

22. Discard supernatant and air dry

23. Dissolve the pellet in 50 ul DEPC H₂O

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Check RNA quality by electrophoresis

V= 5uL DEPC H₂O + 2uL formaldehyde dye + 2uL RNA

Loading to the gel 1%

SỬ DỤNG KIT

Spectrum Plant Total RNA kit

Note: all reagents are already to use!

1. Lyse Tissue Sample

- Grind tissue sample (100mg) into fine powder

- Add 500ul of the Lysis Solution/2-ME Mixture to powder and vortex and vigorously for at least 30s

- Incubate sample at 56⁰C for 3-5 min

2. Pellet Cellular Debris: Centrifuge the sample at max speed for 3 min

3. Filter lysate

- Pipette the lysate supernatant into a Filtration column (blue retainer ring) seated in a 2-ml collection tube. Avoid taking the pellet.

- Close the cap and centrifuge at max speed for 1 min to remove residual debris

- Save the clarified flow-through lysate!!!

4. Bind RNA to column

- Add 250ul Binding Solution to the clarified lysate and mix immediately and thoroughly by pipetting at least 5 times. Do not centrifuge.

- Bind RNA: pipette the mixture into Binding Column (red retainer ring) seated in a 2-ml collection tube. Close the cap and centrifuge at max speed for 1 min.

- Decant flow-through liquid and tap the collection tube (upside down) briefly on clean tissue paper to drain residual liquid.

- Return the column to the collection tube.

5. Column Washes:

1^st wash

- Add 500ul Wash Solution 1 into the column. Close cap and centrifuge at max speed for 1 min

- Decant the flow-through and tap collection tube (upside down) briefly on clean tissue paper then return column to collection tube

2^nd wash

- Add 500ul diluted Wash Solution 2 into column. Close cap and centrifuge at max speed for 30s.

- Discard the flow-through liquid and tap collection tube (upside down) on clean tissue paper for draining residual liquid. Return column to collection tube.

3^rd wash

- Add 500ul diluted Wash Solution 2 into column. Close tap and centrifuge at max speed for 30s.

- Discard the flow-through and tap collection tube (upside down) on clean tissue paper for draining residual liquid. Return column to collection tube.

6. Dry Column

- Centrifuge column at max speed for 1 min to dry.

- Carefully remove the column-tube assembly from the centrifuge to avoid splashing the residual flow-through liquid on the dried column. If the residual flow-through does accidentally contact the column, re-centrifuge for 30s.

7. Elution RNA

1^st elution

- Transfer the column to a new, clean 2-ml collection tube

- Add 50ul Elution Solution directly onto the center of the binding matrix.

- Close cap and let the tube sit for 1 min.

- Centrifuge at max speed for 1 min to elute.

Note: purified RNA is now in the flow-through eluate and ready for immediate use or store at -20⁰C (short term) or – 80⁰C (long term)

2^nd elution (optional).

If the expected RNA yield is >20mg, an additional 10-30% of RNA yield may be recovered from the column with the 2^nd elution.

- Transfer the column to a new, clean 2-ml collection tube.

- Add 50 ul Elution Solution directly onto center of the binding matrix

- Centrifuge at max speed for 1 min to elute.

 Note: purified RNA is now in the flow-through eluate and ready for immediate use or store at -20⁰C (short term) or – 80⁰C (long term)