RNA isolation from Nostoc punctiforme

 

Ref: Meeks lab protocol, Qiagen RNeasy Mini Handbook

 

  1. Harvest ~100 ml non-stationary phase cells (≤ 3 mg/ml ChlA; ~ 300 mg total) in 50 ml conical vials for 5 min in clinical centrifuges at max speed.

 

  1. Pour off supe until traces of pellet move towards mouth of tube (~1-3 ml left).

 

  1. Centrifuge for another 2 min.

 

  1. Pipet 1 ml off bottom of tube into 2 µl Mini BeadBeater screw-cap tubes.

 

  1. Microfuge for 2 min at max speed (12,000 rpm, as for rest of protocol).

 

  1. Remove supe except for 500 µl.

 

  1. Snap freeze the pellet in liquid nitrogen.

 

  1. Store at -80°C until ready to isolate RNA.

 

  1. Add in this order to the still-frozen tube:
    1. 0.58 g 0.5 mm zirconium/silicate beads (Biospec)
    2. 33.3 µl water
    3. 167 µl 3% Celite (shake well before use, stored at -20°C)
    4. 583 µl Tris-buffered phenol (color must be clear, store under argon 4°C)

 

The cell sample should still be frozen when you put it onto the bead beater.  This will ensure that as the cells thaw and break that nucleases are immediately inactivated by phenol.  Shake at high speed, 160 sec on MiniBead Beater (Biospec). Keep samples on ice until all samples are ready.

 

  1. Spin 15 min at 4°C. Meanwhile aliquot 600 µl chloroform to new tubes.

 

  1. Transfer the ~650 µl aqueous (top) layer from cell extract into tubes containing chloroform and mix well by shaking. Can extract a second time for a slightly higher yield, if desired.

 

  1. Spin 10 min at 4°C. Meanwhile, aliquot 500 µl precipitation solution (4 M lithium chloride, 20 mM Tris, pH 7.4, 10 mM EDTA, pH 8) into new tubes.

 

  1. Transfer 500 µl aqueous layer from cell extract into tubes containing precipitation solution, mix by inversion and precipitate at -20°C for 1 hour to O/N (will freeze).

 

  1. Put samples in the centrifuge while it is still frozen.  Spin 15 min at 4°C. Discard supernatant.  Sample must be frozen at the beginning of the spin or you will not get a white RNA pellet.

 

  1. Add 1 ml 70% EtOH and resuspend by pipetting. Spin 10 min at 4°C.  Sample may become translucent at this point.

 

  1. Remove supernatant. Spin 1 min and remove residual supernatant. Resuspend wet pellet in 100 µl TE buffer (RNA-only stock).

 

  1. Add 350 µl Buffer RLT (with BME added at 10 µl/1 ml).  Prepare this mix fresh.

 

  1. Add 250 µl 100% EtOH and mix thoroughly by pipetting. A white precipitate may form at this step; apply it to the column.

 

  1. Apply the sample to an RNeasy mini column inside collection tube. Centrifuge for 15 sec at room temp, as for all subsequent steps. Discard flow-through*.

 

  1. DNase treatment:
    1. Pipet 350 µl Buffer RW1 into RNeasy mini column, centrifuge for 15 sec to wash. Discard flow-through.
    2. Add 10 µl DNase 1 stock solution to 70 µl Buffer RDD. Mix the tube by inversion only.  See below regarding DNaseI preparation.
    3. Pipet DNase I incubation mix directly onto the surface of the membrane of the column and place on benchtop for 15 min.
    4. Pipet 350 µl Buffer RW1 into the RNeasy mini column and centrifuge for 15 sec. Discard flow-through*. Move the column to a new collection tube.

 

  1. Pipet 500 µl Buffer RPE onto the RNeasy column. Centrifuge for 15 sec. Discard flow-through.

 

  1. Add another 500 µl RPE to the column. Centrifuge for 2 min to dry the column.

 

  1. To elute, transfer the column to a new 1.5 µl collection tube. Pipet 25 µl of TE pH 7.4 directly onto the RNeasy silica-gel membrane. Let sit 2 min. Centrifuge for 1 min at max speed to elute. Repeat with fresh TE.  You can also elute with RNase-free ddw also depending on what downstream applications require.  However, yields are generally lower when eluting with ddw vs. TE.

 

  1. Store at -80°C.

 

 

Quantitation of total RNA:

    1. Dilute 1/100 in water (blank with 1% TE in water)
    2. For mg/ml of RNA: Abs 260 * dil factor * 40 mg/ml * 0.001 ml/ml.

Determining RNA quality:

    1. Dilute 1/100 in TE pH 7.4. The Abs 260/ Abs 280 ratio should be between 1.8 and 2.1. The Abs 260/230 should be ~ 2.0, which indicates a lack of contamination from reagents used during RNA isolation.

 

DNaseI preparation:

      a.  RNase-free DNase set (cat. # 79254 from Qiagen) comes lyophilized.  To prepare stock DNase I solution before use, add 550 µl of RNase-free water (provided in the kit).

      b.  Mix gently by inverting the tube, DO NOT VORTEX as the enzyme is especially sensitive to physical denaturation.

      c.  For long term storage, aliquot into single-use volumes.  Make sure to date each tube.  Store at -20oC for up to 9 months.  Thawed aliquots can be stored at +4oC for up to 6 weeks.  DO NOT REFREEZE the aliquots after thawing.

 

Note:  Qiagen RNA column capacity is 100 µg and RNA>200 bases bind to the silica-gel membrane.

 

*Buffers RLT and RW1 are incompatible with bleach.