Hi, I extract RNA from different plant tissues using Trizole as well as Qiagen RNAEasy kit but when ever i extract RNA by these two methods my RNA always degrade and have genomic DNA traces. Please help me what should i do. My protocol for trizole is as follow:
HOMOGENIZATION:
1. Prepare 1.5ml Safe-lock eppendorf tubes and add 5-6 glass-beads.
2. Add 4-6 young leave or (any tissue) to each tube and freeze immediately in liquid nitrogen.
Samples can be used immediately or stored for up to several months at -80°C.
3. Cool these tubes in liquid nitrogen for 5-15 min.
4. Homogenize the tissue in a Geno grinder or mortar and pestle for at least 5 minutes to make it completely crushed.
RNA extraction:
5. Keep out tubes one by one from liquid nitrogen and immediately add 1ml (1000 ul) of TRIZOL to the homogenized tissue and mix it very well for 1 min by vortexing.
6. Incubate at Room Temperature for 5 min.
7. Centrifuge the samples at 12000 rpm for 10min at 4°C.
8. Transfer the supernatant to new RNase free tubes.
9. Add 0.2ml (200 ul) of trichloro methane (chloroform).
10. Shake vigorously by hand or vortex it for 30 seconds.
11. Incubate at RT for 3 min.
12. Centrifuge the samples at 12000 rpm for 10min at 4°C for phase separation.
13. Transfer the aqueous upper phase to new tubes from other three phases in tube.
14. Precipitate RNA by mixing with 0.5 ml (500 ul) isopropanol per 1ml TRIZOL and gently mix by hands.
15. Incubate at RT for 30 minutes OR keep it at -20°C for two hours OR keep it O/N at -20°C.
16. Centrifuge the samples at 13000 rpm for 10min at 4°C to obtain pellets
RNA wash:
17. Remove supernatant and wash pellets with 1ml 75% EtOH (diluted with
DEPC treated water).
18. Vortex once and centrifuge at 7500 x g for 5min at 4°C
19. Discard supernatant and dry pellets for 5 min at RT (or in speed-vac)
20. Dissolve pellets in DEPC treated water
21. Incubate at 55°C for 10 minutes.
Store at –80°C until use.
1. Your glass bead,leaf, homogenizer, grinder, motor pestle should be nuclease free.
2. Your protocol looks good but few thing you have to do carefully, first of all had you observed pink color after step-10, After step -14 there should be a white ring between two solution interface, don't shake too much after step 14 and wait for atleast 2-3 minutes at RT to react, for precipitation of RNA increase the time atleast 2-3 hrs., After step-17 remove the residual ethanol by 10uL pipet tips,don't do step-18 and also don't use speedvac to dry the pellet and dissolve the pellet in RNA storage solution or in TE (pH-7.2-8).
I extract RNa from rat tissue, and i don't freeze it first.. i just put the tissue in a morter tube ( it's kinda a 15 ml tube, but made of glass), add 500 ul TRIZOL and start the homogenezation for 5 min. Then i add another 500 ul in a new RNAse free tube to complete 1 ml TRIZOL and then i do the same steps as you (except 7, 8, 21 and for 15 i incubate for 5-10 min). I get very good RNA, with 260/280 up to 1,95.
I know my tissue is different, but since the cartilage is stroger, thouger tissue than leaves, i supose you could try the same steps as me. (sorry for bad english haha i'm from Argentina).
do you treat your glassware and plastic wares with DEPC... if not this could help you out... trear all your plastic wares even pipette tip, micro centrifuge tube, gloves , etc with DEPC.
And regarding to DNA contamination in RNA try to be less greedy while taking the RNA containing phase(aqueous phase) after centrifugation...
All the best
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