Protocols_RNA Extraction Protocol - BackmanLab/BackmanLab.github.io GitHub Wiki

Procedure: Total RNA Extraction

SOP Prepared by: Emily Pujadas Version: 1.0

  1. Scope

The purpose of this SOP is to describe how to extract RNA from cells in preparation for RTq-PCR Experiments.

  1. Description

This protocol aims to describe the procedure of extracting RNA using the RNeasy Mini Kit (Qiagen). This should be used in conjunction with the RNase-Free DNase Set (Qiagen) to eliminate genomic DNA contamination. Following this protocol, the user should next refer to the RNA Quantification/ Quality Control, Reverse Transcription (cDNA Synthesis), and qPCR Validation (qRT-PCR) Protocols.

  1. Personal Protective Equipment
  • Gloves
  1. Potential Hazards + Safety Precautions

  • Nitrile gloves should be used at all times.

  • RNaseZap RNase Decontamination solution should be used to ensure an RNase-free environment. This can be used for cleaning work surfaces, pipettors, and equipment that must be RNase free.

  1. Materials and Reagents
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  1. Equipment
  • Centrifuge (Tissue Culture Room)
  1. Materials

  • RNaseZap RNase Decontamination Solution (*Invitrogen, Cat# AM9780

    -- Stored at RT in Hi-C room)*

  • RNeasy Mini Kit 250 *(Qiagen, Cat# 74106 -- Stored at RT in Tissue

    Culture Room)*

  • Pipettes and tips

  1. Reagents and Solutions

  • 96-100% Ethanol (as indicated on the bottle of Buffer RPE)

  • 70% Ethanol

  • PBS

  • Trypsin

  • Beta-mercaptoethanol *(Thermo Fisher Scientific, Cat# 21985023 --

    Stored at RT)*

= Total RNA Extraction (Cont.) =

  1. Procedure

Things to do Before Starting:

  1. Add B-mercaptoethanol to Buffer RLT before use.
  • Add 10 ul B-ME per 1 ml Buffer RLT. Dispense in a fume hood and wear appropriate clothing. Buffer RLT containing B-ME can be stored at room temperature for up to 1 month.

NOTE 1: When working with RNA, care must be taken to avoid degradation by RNases, which are extremely stable and active. Intracellular RNases are released during the lysis step of the RNA isolation procedure and must be rapidly and thoroughly inactivated to obtain high-quality RNA. Beta-mercaptoethanol  (B-ME) is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds and destroying the native conformation required for enzyme functionality. In combination with the strong, but temporary denaturing effects of guanidinium isothiocyanate (GITC) contained in buffer RLT of the RNeasy Kits, any RNases present in the material to be extracted from will be completely inactivated. 

  1. Buffer RPE is supplied as a concentrate. Before using for the first

    time, add 4 volumes of ethanol (96-100%) as indicated on the bottle to obtain a working solution.

  2. Prepare DNase I stock solution.

    • (see eliminating genomic DNA contamination section)

RNA Extraction (Part 1):

NOTE 2: Perform an RNA extraction for non-transduced cells as a [negative control]{.ul}

  1. Harvest cells

  • Start with no more than 3-4 x 10^6^ cells (counting cells is the most accurate way to quantitate the amount of starting material)

  • To trypsinize and collect cells:

    • Determine the number of cells.

    • Aspirate the medium, and wash the cells with PBS.

    • Aspirate the PBS, and add 0.1-0.25% trypsin in PBS. After the cells detach from the dish/ flask, add medium (w/ serum to inactivate trypsin), transfer the cells to an RNase-free glass or polypropylene centrifuge tube and centrifuge at 300 x g for 5 min.

    • Completely aspirate the supernatant.

NOTE 3: Incomplete removal of cell-culture medium will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the RNeasy membrane. Both effects may reduce RNA yield.

  1. Disrupt the cells by adding Buffer RLT.

  • For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add the appropriate volume of Buffer RLT.

    • For <5 x 10^6^ pelleted cells, add 350 ul of Buffer RLT

    • For 5 x 10^6^ - 1 x 10^7^ pelleted cells, add 600 ul of Buffer RLT

  • Vortex to mix.

  1. Homogenize the lysate
  • Pipet the lysate directly into a QIAshredder spin column placed in a 2 ml collection tube, and centrifuge for 2 min at full speed.
  1. Add 1 volume of 70% ethanol to the homogenized lysate, and mix well by pipetting. Do NOT centrifuge

NOTE 4: The volume of lysate may be less than 350 ul or 600 ul due to loss during homogenization.

NOTE 5: When purifying RNA from certain cell lines, precipitates may be visible after addition of ethanol. This does not affect the procedure.

  1. Transfer up to 700 ul of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at 8000 x g (10,000 rpm). Discard the flow-through.

NOTE 6: The flow-through contains Buffer RLT and is therefore not compatible with bleach.

[NOTE 7:]{.ul} Reuse the collection tube.

[NOTE 8:]{.ul} If the sample volume exceeds 700 ul, centrifuge successive aliquots in the same RNeasy spin column. Discarded the flow-through after each centrifugation.

Eliminating Genomic DNA Contamination:

[NOTE 9:]{.ul} Before starting,

  • Prepare DNase I stock solution before using the RNase-Free DNase Set for the first time. Dissolve the lyophilized DNase (1500 Kunitz units) in 550 ul of the RNase-free water provided. To avoid loss of DNase I, do not open the vial. Inject RNase-free water into the vial using an RNase-free needle and syringe. Mix gently by inverting the vial. 

  • For long-term storage of DNase I, remove the stock solution from the glass vial, divide it into single-use aliquots, and store at -20 C for up to 9 months. Thawed aliquots can be stored at 2-8 C for up to 6 weeks. Do not refreeze the aliquots after thawing.

  1. Add 350 ul Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 8000 x g (10,000 rpm) to wash the spin column membrane. Discard the flow-through.

NOTE 10: The flow-through contains Buffer RW1 and is therefore not compatible with bleach.

NOTE 11: Reuse the collection tube in Step 4.

  1. Add 10 ul DNase I stock solution to 70 ul Buffer RDD. Mix by gently inverting the tube, and centrifuge briefly to collect residual liquid from the sides of the tube.

NOTE 12: DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube. Do NOT vortex.

  1. Add the DNase I incubation mix (80 ul) directly to the RNeasy spin column membrane, and place on the benchtop (20-30 C) for 15 min.

NOTE 13: Be sure to add the DNase I incubation mix directly to the RNeasy spin column membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or O-ring of the spin column.

  1. Add 350 ul Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 8000 x g (10,000 rpm). Discard the flow-through. Continue with the first Buffer RPE wash step in the relevant protocol.

NOTE 14: The flow-through contains Buffer RW1 and is therefore not compatible with bleach.

RNA Extraction (Part 2):

  1. Add 500 ul Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 8000 x g (10,000 rpm) to wash the spin column membrane. Discard the flow-through.

NOTE 15: Reuse the collection tube in Step 11 (next step).

NOTE 16: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use (See "Things to do before starting)

  1. Add 500 ul Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at 8000 x g (10,000 rpm) to wash the spin column membrane.

NOTE 17: The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions.

NOTE 18: After centrifugation, carefully remove the RNeasy spin column so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.

  1. Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-throw. Close the lid gently, and centrifuge at full speed for 1 min.

NOTE 19: Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin column after step 11.

  1. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30-50 ul RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at 8000 x g (10,000 rpm) to elute the RNA.

  2. If the expected RNA yield is > 30 ug, repeat step 13 (last step) using another 30-50 ul RNase-free water, or using the eluate from step 13 (if high RNA concentration is required). Reuse the collection tube from step 13.

NOTE 20: If using the eluate from step 10, the RNA yield will be 15-30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.

  1. Disposals | Spills | Incidents

Waste (see waste segregations and collection SOP for complete information)

  • All waste containers should be closed once ¾ 's and placed into the appropriate location for pick up full.

  • All waste containers should be properly labelled with the following information:

    • PI's name

    • User | Responsible person

    • Content

    • Waste class (e.g. chemical, biohazard, radioactive, etc.)

  • = Total RNA Extraction (Cont.) =

    • Waste type (e.g. sharps, plastic, glass, metal, etc.)

    • Waste "status" (e.g. liquid, solid, etc.)

[Protocol Specific]{.ul}

  1. Waste

    • RNase-free Needle -- place in a puncture resistant container

      with a "Chemically contaminated waste" labelled

    • Beta-mercaptoethanol -- place in the bottle in the fume hood

      that specifically states "beta-mercaptoethanol waste"

    • Empty bottles of EtOH -- remove or unutilized the label, and

      mark as "rinsed to dispose"

    <!-- -->

    • Rinse 3x, leave open in the fume hood to dry

    • Place by/ in the regular waste bins for collection with regular waste

    • Biological samples -- inactivate with 10% bleach for 30 minutes and discard in lab sink

  2. Spills | Incidents:

Considering the type of reagents used (EtOH) and sample's volume (less than 10 mL), the risk of hazardous spills is minimal to non-existent.

  1. References

Directions for using the cell counter:

https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/cellanalysis/Files/0914/MAN0010644_Countess%20II.pdf

= Total RNA Extraction (Cont.) =

SUMMARY FLOWCHART OF PROCEDURES TO CONSIDER IN CASE OF A

xxxx EMERGENCY

[Only when applicable].

**
**

= Total RNA Extraction (Cont.) =

// Protocol Summary//

  1. Harvest cells & determine the amount of starting material

  2. Disrupt the cells by adding Buffer RLT & vortex to mix

  3. Homogenize the lysate

  4. Add 1 volume of 70% EtOH and mix well by pipetting

  5. Transfer up to 700 ul of the sample to an RNeasy spin column placed

    in a 2ml collection tube. Discard the flow through.

  6. Add 350 ul Buffer RW1 to the RNeasy spin column. Centrifuge 15 s at

    8000 x g (10,000 rpm) to wash the spin column membrane. Discard the flow-through.

  7. Add 10 ul DNase I stock solution to 70 ul Buffer RDD. Mix by gently

    inverting.

  8. Add the DNase I incubation mix (80 ul) directly to the RNeasy spin

    column membrane, and place on the benchtop (20-30 C) for 15 min.

  9. Add 350 ul Buffer RW1 tot eh RNeasy spin column. Centrifuge 15 s at

    8000 x g (10,000 rpm) to wash the spin column membrane. Discard the flow-through.

  10. Add 500 ul Buffer RPE to the RNeasy spin column. Centrifuge 15 s at

    8000 x g (10,000 rpm) to wash the spin column membrane. Discard the flow-through.

  11. Add 500 ul Buffer RPE to the RNeasy spin column. Centrifuge 2 min at

    8000 x g (10,000 rpm) to wash the spin column membrane.

  12. Place the RNeasy spin column in a new 1.5 ml collection tube. Add

    30-50 ul RNase-free water directly to the spin column membrane. Centrifuge for 1 min at 8000 x g (10,000 rpm) to elute the RNA.

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