Saturday, October 24, 2009

Sherman: Molecular work

RNA extraction and quantification

The method of extracting and purifying RNA, which will be used for molecular studies

It involves harvesting cells, disrupting cells, homogenizing cells, bind RNA, collect RNA and wash membrane


Materials

  1. Rneasy-free pipette tips (filter tips)
  2. Microcentrifuge
  3. 96-100% and 70% non-denatured EtOH
  4. QIA shredder spin column
  5. Rnase spin column
  6. Pipette
  7. Micropipette
  8. Falcon tube
  9. Light microscope
  10. Counter
  11. RNA extraction kit
    1. RLT
    2. RW1
    3. RPE
  12. MQ water
  13. Cuvettes
  14. Spectrophotometer
  15. Centrifuge


Method

  1. Harvest cells (trypsinisng with treatment & transferring into a falcon tube for centrifuge)
  2. Centrifuge at 600rpm for 5mins, remove supernatant and count cells
  3. Centrifuge the remaining cells and remove the supernatant
  4. Disrupt the cells; on the cell pellets, add 350ul of RLT if the cell count is <>6 cells, or 600ul of RLT for 5 x 106-107. Mix well (an alternative is to vortex for 1min)
  5. Homogenise cells; pipette lysate into QIA shredder spin column in 2ml collection tube (the shredder spin column will cut and lyse the cells more evenly to allow higher RNA yield)
  6. Centrifuge for 2mins at full speed and discard column (the RNA is in the flow-through)
  7. Bind RNA; add 1 volume of 70% EtOH to lysate (flow-through) and mix by pipetting - to precipitate the RNA

* 1 volume = amount of RLT that was added at the given cell count

  1. Collect RNA; transfer 700ul of sample into Rnase spin column in 2ml collection tube - RNA will be trapped in the membrane of the Rnase spin column
  2. Centrifuge for 15secs at full speed. Discard the flow-through
  3. Wash membrane; add 700ul of RW1 to the spin column, repeat the step above
  4. Add 500ul of RPE to the spin column, repeat the step above
  5. Repeat the step above, but centrifuge for 2mins (dry the RNA)
  6. Elute RNA; Transfer the spin column into an eppendorf tube
  7. Add 30ul of Rnase-free water (directly in the middle of the membrane, without touching it)
  8. Centrifuge at max speed for 1min
  9. Repeat the step above, recycling the Rnase-free water. This is to get higher yield of RNA
  10. Collect the flow-through.
  11. Label 5 baby eppendorf tubes for the storage of RNA (stored at -80ºc)
  12. Aliquote 5ul of RNA into the first 4 tubes, and 10ul into the last tube for RNA quantification

RNA quantitation

An absorbance of 1 unit at 260nm corresponds to 40ug of RNA per ml

Aλ260 = 1, concentration = 40ug/ml. To ensure significance, reading should be greater than 0.15

  1. Pipette 2ul of RNA into a cuvette and 498ul of MQ water

*Ensure that the RNA is mixed with the MQ water, otherwise there will be little yield reading

  1. Set up a blank sample (500ul MQ water) and the diluted RNA sample
  2. Insert the cuvettes into the spectrophotometer
  3. Set the wavelength (A260 and A280)
  4. Set reference for the blank to 0.000 absorbance
  5. [RUN] samples according
  6. Take readings for each cell line at the 2 given wavelengths (A260 and A280)

Purity of the RNA is measured by dividing the wavelength at A260 by A280. A ratio of 1.8 - 2.0 will give a pure result
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7 comments:

  1. TG01-Group 2October 27, 2009 at 9:39 PM

    Hey Sherman!

    what if RNA quantitation fails? what is the possible cause? how do you rectify it?

    Thanks!

    Yeo Sok Kian Jocelyn
    0703359J

    ReplyDelete
  2. MedScientists of Grp 6October 27, 2009 at 9:44 PM

    yo sherman!

    how do you harvest the cells? what kind of trypsin do you use?

    thank you very much!

    Joanna Yeo!
    0702054H

    ReplyDelete
  3. The Madtechs Gp09 - TG02October 28, 2009 at 6:45 PM

    Hi sherman!!

    For step 8 (collect RNA), you said "transfer 700ul of sample into Rnase spin column in 2ml collection tube", so the sample refer to precipitated RNA? (because in step 7, you precipitate RNA)

    And continue from step 8, "RNA will be trapped in the membrane of the Rnase spin column", so the RNA is the precipitated one or the pure one?

    And what is the purpose of precipitating RNA? Wont it contaminate the RNA?

    Sry, i'm quite confused.

    Thks!

    Zhang'e
    0704086H
    TG02

    ReplyDelete
  4. MedTech JrOctober 28, 2009 at 10:57 PM

    Hi Jocelyn!

    if quantitation fails, we'll just have to redo, or troubleshoot the procedures.

    The underlying causes can be the technique of quantitation, nucleic contamination, contaminated eppendorf tubes, degraded 70% ethanol.

    To rectify the problem,

    #1. Autoclave the eppendorf tubes
    #2. Change the 70% ethanol
    #3. Ensure clean handling of RNA product

    For technique wise, do the following 2 methods;

    #1. Aliquote the RNA and MQ water into a separate RNA-grade eppendorf tube and pulse/spin before transferring into a cuvette

    #2. Aliquote the RNA and MQ water into the cuvetter directly. Parafilm the cuvette and pulse/spin


    Apart from that...a spectrophotometer is considered an "old technology" to quantitate RNA...rather it'll be better to use a NanoDrop

    ReplyDelete
  5. MedTech JrOctober 28, 2009 at 11:02 PM

    Hi Joanna! =)

    This harvesting is exclusive for this research's Primary Cell Lines

    Harvesting cells:

    1. Ensure the cells are confluent under light microscopy.
    2. Suck out all of the media using a vacuum
    3. Treat the cells with PBS for 5sec
    4. Remove the PBS
    5. Pipette 5.5ml of Trypsin/PBS for 10sec
    6. Remove 5ml of Trypsin/PBS and leave it for 5mins
    7. Pipette 2ml of Cyto Mixed Media
    8. Centrifuge at 600rpm for 5mins at 4degrees celcius
    9. Remove the supernatant
    10. RNA extraction step starts here


    The trypsin used is Trypsin/PBS


    Thanks for the question!

    Sherman Lim
    0703326I

    ReplyDelete
  6. MedTech JrOctober 28, 2009 at 11:09 PM

    Hi Zhang'E!

    The RNA that is transferred into the Rneasy Spin Column is precipitated RNA

    The RNA that is trapped in the membrane is just RNA.

    The aim of the 70% EtOH is to precipitate the RNA so that it's MW is big enough to be trapped in the membrane...
    Pure RNA is no different from precipitated RNA. It's still RNA.
    Then after that there's the washing step to remove the precipitation so that you'll ultimately get purified RNA.

    Hope it answers your question =)

    ReplyDelete
  7. jenashling-shamunaNovember 1, 2009 at 1:17 AM

    Hi sherman. Just to be sure, when you say centrifuge the remaining cells and discard the supernatent, the remaining cells mean the cells in the first supernatent after the first centrifuge?
    And during the disrupting of the cells step, the cells are still not lyse yet right so why do we add the RLT( RNA extraction kit) before the cells are lysed?

    ReplyDelete

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