5.4 RNA Extraction

Submitted by margaret on Wed, 11/16/2011 - 18:32

Need a housekeeping gene

Need a better way to estimate RNA concentration. Forgo spectrophotometer? Just do gel? Get RNA ladder? Or is an estimate (or fold-difference) sufficient to get comparable amounts of RNA into each RT reaction? The absolute amounts don't really matter, do they?

Nanodrop? calibrate once with water (1 min very fast.) sample. type in name. 1.7 µL sample, run.

Replicates? allow 4 per group instead of 2? Require groups with same gene to work together? Either RNA extraction or RT is too touchy - if they had more replicates, would they be more likely to get usable data?

Stay only in the 3` end. You seldom get a full size transcript. Add this to the criteria for primer design.

2015

  • Use ball mill to grind samples
  • Re calculate for up to 9 extractions per group

W 4/11/12

Prepare total RNA from selected Arabidopsis tissues and accurately quantify RNA concentration and yield.

Equipment:

  • Balance + small weigh boats
  • Liquid nitrogen & ice buckets
  • Blue pestles1 & matching tubes (individually wrapped in kits drawer)
  • 2 Lilac and 2 pink tubes from the RNEasy2 kit
  • 8 2mL collection tubes (from kit)
  • 2 capless 2 mL tubes, sterilized
  • brand-new, untouched tips (with warning labels: RNAse free! Gloves only!)
  • brand-new, untouched microfuge tubes (with warning labels)

Reagents

One tube for each group (2 per group of RPE), plus 5 to have on hand

  • 1 mL RLT (450 µL x 2)
  • 1 mL 100% ethanol (225 µL x 2)
  • 1.5 mL RW1 (350 µL x 4)
  • 200 µL RDD (70 µL x 2)
  • 10 µL DNase3 aliquot all of it, freeze. Label tube “10µL DNase”
  • 3 mL RPE (500 x 4) 2 tubes per group, each 1.5 mL
  • 200 µL RNase-free H2O (50 + 10 + 5 µL)
  • 1.5 mL TrisHCl 10 mM pH 7.5 (90 + 100 µL) freshly filtered
  • 100 µL new loading dye (i.e., not contaminated with RNase) (The loading dye that comes with Fermentas ladders is good)

Follow kit instructions for adding β-M-EtOH

1.0% agarose – enough for 12 groups + 2 accidents

HMW ladder. remove the LMW ladder from their boxes; replace with HMW (in Fermentas box in freezer)

Full carboy of 1X TAE

Remove anything that might be contaminated with RNAse, e.g.

  • water
  • dilution buffer
  • Loading dye
  • Opened tips and tubes containers

Order the DNase separately. Qiagen 79254.

The kit comes with RNase-free water and RDD for diluting the DNAse

To program the Spectrophotometer:

  • Main Menu
    • RNA
    • Setup
    • Dilution 0010 (µL RNA) + 0090 (µL diluent)
    • Factor 40
    • Units µg/mL
    • Resolution= 0.001 (max)
    • λ (wavelength) = 260

Save RNA samples in the -80!

  1. Disposable Pellet Pestle without microtubes; 1.5mL K749521-1500 Kimble Chase Kontes Case of 100 for $54.74 ↩︎

  2. RNeasy Plant Mini Kit (50) Qiagen 74904 ↩︎

  3. DNase I Qiagen 79254 http://www.qiagen.com/products/accessories/rnase-freednaseset.aspx
    Inject 550µL RNAse-free water into the DNAse vial. Make 10 µL aliquots. Need ~48 labels. Freeze. Give away the excess. Can extend slightly by adding 575 µL to get 57 aliquots per vial instead of 55. ↩︎