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Concentrating RNA

Concentrating RNA - RNA Forum

Concentrating RNA - RNA Forum. Discuss RNA including mRNA structure and function, rRNA, tRNAs, and the biochemistry of other RNA molecules.


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  #1  
Old 05-14-2010, 06:28 AM
Pipette Filler
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Default Concentrating RNA



Hi all,

I have a few samples of RNA that I want to use for RACE PCR. The concentration in the samples is too low (around 30-60ng/microlitre) to make RACE cDNA at the moment but I think I can get a high enough concentration if I pool my samples (I have about 30 microlitres from several RNA extractions) and concentrate them down to about 8 microlitres. I am just wondering what the best method for concentrating the RNA. We have a speed vac in the building, but I think it only runs at ambient temperature at the best so I am a bit worried about degrading the RNA. Ive also considered ethanol precipitation but I'm worried about losing some RNA through that method. Ive had another suggestion that I can leave the sample in a fume hood and just let the water evaporate but that sounds a bit risky also. What is the safest method to concentrate the RNA?
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Old 11-01-2010, 10:16 PM
Pipette Filler
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Default Re: Concentrating RNA

I don't know if this will help but in our lab when we concentrate RNA we use a Speed-Vac at 60 oC and dry for about 15 mins per 10 ul of RNA. The RNA is then fine for cDNA preparation and qPCR so I doubt the stability is being affected!

Hope that helps!
William
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Old 11-02-2010, 03:31 PM
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Default Re: Concentrating RNA

The original poster has probably graduated by now...
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Old 11-02-2010, 04:51 PM
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Default Re: Concentrating RNA

Oops did not even notice that! Tho it may help someone else so alls well!
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Old 12-09-2010, 04:56 PM
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Default Re: Concentrating RNA

Quote:
Originally Posted by Wandrews963 View Post
I don't know if this will help but in our lab when we concentrate RNA we use a Speed-Vac at 60 oC and dry for about 15 mins per 10 ul of RNA. The RNA is then fine for cDNA preparation and qPCR so I doubt the stability is being affected!

Hope that helps!
William
This never occured to me to try. Definately worth remembering.

Thanks for the Tip!
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Old 10-14-2014, 10:27 AM
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Smile Re: Concentrating RNA

Mentor Goldern VivienLauYW
RNA Extraction (“Invitrogen” TRIZOL Reagent #15596-018)
1. Add 3ml TRIZOL reagent in 40x16um frozen sections.
2. Mix and incubate at room temperature for 5 minutes.
3. Add 600ul chloroform to the sample. Shake vigorously by hand for 15 seconds and incubate at room temperature at room temperature for 3 minutes.
4. Spin at 12000rpm at 4˚C for 10 minutes.
5. Transfer the aqueous phase to the new tube.
6. Precipitate the RNA by mixing with 1.5ml isopropyl alcohol. Incubate at room temperature for 10 minutes.
7. Spin at 12000rpm at 4˚C for 10 minutes.
8. Wash the RNA pellet with 3ml 75% ethanol. Mix.
9. Spin at 7500rpm at 4˚C for 5 minutes
10. Air dry the pellet.
11. Dissolve RNA in DEPC H2O.
No mAb (-ve) cell line, check AutoF.
If –ve stained, continue keep it in NGS; If +ve stained, take out and end it by replace solution.

DNase Treatment of RNA
1. Prepare the reaction mixture: 10X DNase buffer 6ul, DNase I (10U/ul (2ul, RNase Out (40U/ul) 1ul, 10ug RNA + DEPC H20 51ul
2. Incubate at 37˚C for 30 minutes
3. Stop reaction by adding 6ul 0.1M EDTA (pH8.0)
4. Add DEPC H20 to 400ul, then add 400ul H20 phenol. Chloroform:IAA (50:49:1)
5. Mix and Spin at 12000 rpm at 4˚C for 10 minutes
6. Transfer the stop phase to 1.5ml eppendorf tube. Repeat steps 4 to 6 once.
7. Precipate RNA by adding 40ul 3M NaOAc and 800ul absolute ethanol.
8. Keep at -20˚C for at least 2 hours.
9. Spin at 12000rpm at 4˚C for 5 minutes
10. Remove supernatant and wash the pellet with 500ul 70% ethanol.
11. Spin at 12000rpm at 4˚C for 5 minutes.
12. Remove supernatant and dry the pellet.
13. Resuspend the RNA pellet in 15ul DEPC H2O.
14. Store at -80˚C.

Reagents
10X DNase buffer 1M Tris-HCL (pH7.5) 400ul, 5M NaCl 20ul, 2M MgCl2 30ul, DEPC H2O) 550ul.

Reverse Transcription (‘Invitrogen’ SuperScript First-Strand Synthesis System for RT-PCR #11904-018)
1. Prepare DNase treated RNA/primer mixtures in sterile 0.5ml tubes as follows: 5ug total RNA + DEPC H2O 8ul, 10mM dNTP mix 1ul, Oligo dT (0.5ug/ul) 1ul.
2. Incubate at 65˚C for 5 minutes, then place on ice for at least 1 minute
3. Prepare the master mix: 10X Room Temperature buffer 2ul, 25mM MgCl2 4ul, 0.1M DTT 2ul, RNase Out (40U/ul) 1ul
4. Add 9ul of reaction mixture to each RNA/primer mixture, mix gently.
5. Incubate at 42˚C for 2 minutes.
6. Add 1ul (50 units) of SuperScript II Room Temperature to each tube, mix, and incubate at 42˚C for 50 minutes.
7. Terminate the reaction at 70˚C for 15 minutes. Chill on ice.
8. Flick spin. Add 1ul of RNase H (use your own set of pipetman) to each tube and incubate at 37˚C for 20 minutes.
9. Add 380ul sterile MQ-H2O to each tube.
10. Store cDNA at -20˚C.

Immunohistochemistry on paraffin section by Envision Kit
1. Cut 4-6um thick paraffin section on Tespa coated slide.
Dewax the paraffin sections in xylene (3 minutes, 3 times) and rehydrate through ethanol series (99% ETOH 3 minutes, 3 times); 95% ETOH 3 minutes and 80% ETOH 3 minutes
3. Rinse with distilled H2O 3 times
4. Antigen retrieval by pressure cooker method at 200˚C. Heat sections in 2L 10mM citrate buffer (pH6.0) for 5 minutes when the pressure reach at maximum.
5. Cool down the sections to Room Temperature.
6. Rinse with distilled H20 3 times
7. Remove endogenous peroxidise activities by treating the sections in 3% H2O2 70ul (63ul + 7ul) in H2O for 10 minutes at Room Trmperature. (Add 15ml 30% H2O in 135ml H2O)
8. Rinse with distilled H2O 2 times, then 1X TBS.
9. Add 1200 diluted primary mouse PTEN antibody (diluted in 1% BSA) to the section and incubate in a moist chamber at 37˚C for overnight.
10. Rinse with TBS 3 minutes for 3 times
11. Add a drop of anti-mouse antibody labelled with polymer+HRP and incubate at room temperature for 30min.
12. Rinse the section with TBS 3 minutes for 3 times
13. Develop the signal in freshly prepared DAB at room temperature. (1 drop DAB in 1ml substrate buffer)
14. Wash in tap-water.
15. Counterstain nuclei with Mayer’s Haemtoxylin for 1 minute.
16. Blue in Scott’s Tap H2O
17. Wash in tap-water, dehydrate, clear and mount.

Reagents:
10X citrate buffer (pH6.0), 1L: Citric acid 21g, NaOH 10.4g
20X Tris buffer saline (TBS) (pH7.6), 1L: Trizma 60.57g, NaCl 87.66g
BSA mix with TBS for blocking
BSA mix with TBS mix with antibody for dilution
Mili = m (10-3), Micro = u (10-6)
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