Best Practices

Best Practices

Handling RNA

RNA is highly susceptible to degradation by RNAse enzymes. RNAse enzymes are present in cells and tissues, and carried on hands, labware, and dust. They are very stable and difficult to inactivate. For these reasons, it is important to follow best laboratory practices while preparing and handling RNA samples:

  • When harvesting total RNA, use a method that quickly disrupts tissue and isolates and stabilizes RNA.
  • Wear gloves and use sterile technique at all times.
  • Reserve a set of pipettes for RNA work. Use sterile RNAse-free filter pipette tips to prevent cross-contamination.
  • Use disposable plasticware that is certified to be RNAse-free. Illumina recommends the use of non-stick sterile RNAse-free microfuge tubes. A set of these tubes should be designated for this protocol and should not be used for other lab work.
  • All reagents should be prepared from RNAse-free components, including ultra pure water.
  • Store RNA samples by freezing.
  • Use a RNAse/DNAse decontamination solution to decontaminate work surfaces and equipment prior to starting this protocol.

Handling Liquids

Good liquid handling measures are essential, particularly when quantifying libraries or diluting concentrated libraries for making clusters.

  • Small differences in volumes (±0.5 µl) can sometimes give rise to very large differences in cluster numbers (~100,000).
  • Small volume pipetting can be a source of potential error in protocols that require generation of standard curves, such as PicoGreen assays or qPCR, or those that require small but precise volumes, such as the Agilent Bioanalyzer.
  • If small volumes are unavoidable, then due diligence should be taken to ensure that pipettes are correctly calibrated.
  • Make sure that pipettes are not used at the volume extremes of their performance specifications.

Handling Master Mix Reagents

  • When handling the master mix reagents minimize freeze-thaw cycles. If you do not intend to consume the reagents in one use, dispense the reagent into aliquots after the initial thaw and refreeze the aliquots in order to avoid excessive freeze-thaw cycles. However, if you aliquot, you might not have enough reagents for the full number of reactions over multiple uses.
  • After thawing, mix the ELM4, PMM2, RCS1 by inverting the tube, then centrifuge briefly (approximately 5 seconds).

Handling Magnetic Beads

Follow appropriate handling methods when working with AMPure XP Beads and OB1:

  • Cleanup procedures have only been validated using the 96-well plates and the magnetic stand specified in the Consumables and Equipment list in the TruSeq Targeted RNA Expression Guide. Comparable performance is not guaranteed when using a microcentrifuge tube or other formats, or other magnets.
  • Prior to use, allow the beads to come to room temperature.
  • Do not reuse beads. Always add fresh beads when performing the procedures.
  • Immediately prior to use, vortex the beads until they are well dispersed. The color of the liquid should appear homogeneous.
  • When pipetting the beads, pipette very slowly and dispense very slowly due to the viscosity of the solution.
  • Pipetting with the tips at the bottom of the well and not pipetting the entire volume of the solution helps prevent the solution from foaming. Excessive foaming leads to sample loss, because the foam is not transferred out of the plate efficiently.
  • After adding the beads to the reaction, seal the plate and shake the plate on a microplate shaker at 1800 rpm for 2 minutes. Repeat, if necessary, until the color of the mixture appears homogeneous after mixing.
  • Take care to minimize bead loss which can impact final yields.
  • Change the tips for each sample, unless specified otherwise.
  • Let the mixed samples incubate at room temperature for the time indicated in the protocol to ensure maximum recovery.
  • When removing and discarding supernatant from the wells, use a single channel or multichannel pipette and take care not to disturb the beads.
  • When aspirating the cleared solution from the reaction plate and wash step, it is important to keep the plate on the magnetic stand and to not disturb the separated magnetic beads. Aspirate slowly to prevent the beads from sliding down the sides of the wells and into the pipette tips.
  • To prevent the carryover of beads after elution, approximately 2.5 μl of supernatant are left when the eluates are removed from the bead pellet.

Follow these appropriate handling methods when working with AMPure XP Beads specifically:

  • Prepare fresh 80% ethanol. Ethanol tends to absorb water from the air, therefore, fresh 80% ethanol should be prepared for optimal results.
  • Be sure to remove all of the ethanol from the bottom of the wells, as it may contain residual contaminants.
  • Keep the reaction plate on the magnetic stand and let it air-dry at room temperature to prevent potential bead loss due to electrostatic forces. Allow for the complete evaporation of residual ethanol, as the presence of ethanol will impact the performance of the subsequent reactions. Illumina recommends at least 15 minutes drying time, but a longer drying time may be required. Remaining ethanol can be removed with a 10 μl pipette.
  • Avoid over drying the beads, which can impact final yields.
  • Do not scrape the beads from the edge of the well using the pipette tip.
  • Resuspend the dried pellets by shaking.
  • To maximize sample recovery during elution, incubate the sample/bead mix for 2 minutes at room temperature before placing the samples onto the magnet.

Avoid Cross-Contamination

  • Open only one adapter tube at a time.
  • Change the tips for each sample, unless specified otherwise.
  • Pipette carefully to avoid spillage.
  • Clean pipettes and change gloves between handling different adapter stocks.
  • Clean work surfaces thoroughly before and after the procedure.

Equipment

  • Review the programming instructions for your thermal cycler user guide to ensure that it is programmed appropriately using the heated lid function.
  • Calibrate the microplate shaker with a stroboscope and set it to 1800 rpm.