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This purpose of this experiment is to isolate high quality RNAs from pollen grains, which lays the foundation for further studies, like gene expression analysis and cDNA cloning.

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[Bio101] RNA Isolation from Arabidopsis Pollen Grains

Plant Science > Plant molecular biology > RNA > RNA extraction

[Abstract] This purpose of this experiment is to isolate high quality RNAs from pollen grains, which lays the foundation for further studies, like gene expression analysis and cDNA cloning.

Disposables and Reagents

 

1.        Mannitol (Fisher/VWR)

2.        Trizol (Invitrogen)

3.        Chloroform (Fisher)

4.        75% Ethanol

5.        Liquid Nitrogen

6.        RNase-free water

7.        50 ml falcon tubes

8.        100 μm nylon mesh

 

Equipments

 

1.        Ceramic mortar and pestles.

2.        Nanodrop (Thermo Scientific)

3.        Centrifuge (Eppendorf)

4.        Vortexer (VWR)

5.        Fume hood

6.        500 ml flask

 

Procedure

 

1.        Pollen collection:

To collect mature pollen grains, stage 13 flowers (Sanders et al. 1999) were used .

1)       Collect flowers and put into a 500 ml flask.  

2)       Add 300 ml ice-cold 0.3 M mannitol.

3)       Hand-shake the flask vigorously for 2 min.

4)       Filter the pollen suspension through 100 μm nylon mesh.

5)       Collect pollen by centrifugation using 50 ml falcon tubes. (450 g, 5 min, 4°C). Repeat this step until finish all the pollen suspension.

6)       Transfer pollen pellet into a 1.5 ml centrifuge tube. You can stop here by storing pollen at -80 oC, or proceed to the RNA isolation steps.

2.        Homogenization:

7)       Put into liquid N2.

8)       Homogenize pollen with mortar and pestles. Try to be as quick as possible at this step.

9)       Add TRIzol reagent (1 ml reagent/ 50-100 mg tissue, the sample volume should not exceed 10% of the volume of TRIzol used for homogenization, as suggested by the TRIzol protocol).

3.        Phase separation

10)   Incubate the homogenized samples for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes.

11)   Add 0.2 ml of chloroform per 1 ml of TRIzol reagent under fume hood. Cap sample tubes securely. 

12)   Shake tubes vigorously by hand for 15 seconds and incubate them at RT for 2 to 3 minutes.

13)   Centrifuge the samples at no more than 12,000 x g for 15 minutes at 4 °C.

14)   Transfer the upper aqueous phase to a fresh tube.

4.        RNA precipitation.

15)   Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol (Use 0.5 ml of isopropyl alcohol per 1 ml of TRIzol reagent used for the initial homogenization).

16)   Incubate samples at RT for 10 minutes.

17)   Centrifuge at no more than 12,000 x g for 10 minutes at 4 °C.

18)   Discard the supernatant.

5.        RNA wash.

19)   Wash the RNA pellet with 75% ethanol (use at least 1 ml of 75% Ethanol per 1 ml of TRIzol reagent used for the initial homogenization).

20)   Mix the sample by vortexing.

21)   Centrifuge at no more than 7,500 x g for 5 mintues at 4 °C.

22)   Discard the supernatant. Now you get the RNA pellet at the tube bottom.

6.        Dissolving RNA

23)   Briefly dry the RNA pellet on bench at room temperature (10~20 min).

24)   Dissolve RNA in RNase-free water by passing the solution in a few times through a pipette tip.

25)   Incubate at 55 to 60 °C for 10 minutes. Tap the tube several times during the incubation.

26)   Use Nanodrop to test the quantity and quality of the RNA.

27)   Store the RNA sample in -80 °C for future use.

 

References

 

1.        Sanders P.M., Bui A.Q., Weterings K., McIntire K.N., Hsu Y.-C., Lee P.Y., Truong M.T., Beals T.P., Goldberg R.B. (1999). Anther developmental defects in <i>Arabidopsis thaliana</i> male-sterile mutants.Sexual Plant Reproduction 11(6): 297-322. 

2.        Honys D., Twell D. (2003). Comparative analysis of the Arabidopsis pollen transcriptome. Plant Physiology 132(2): 640-52.  http://www.ncbi.nlm.nih.gov/pubmed/12805594

3.        Lu Y., Chanroj S., Zulkifli L., Johnson M.A., Uozumi N., Cheung A., Sze H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93. 

 



How to cite: Lu, Y. (2011). RNA Isolation from Arabidopsis Pollen Grains. Bio-protocol Bio101: e67. DOI: 10.21769/BioProtoc.67; Full Text



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