protocol_35.txt

Making spike-in transcripts for mRNA normalization
Grow DH5alpha ecoli cells overnight with pSP64 poly(A) plasmid containing inserted spike-in sequence.
Miniprep plasmids, quantify.
You'll need a lot of plasmid.
Set up the digestion reaction in 50ul 1x EcoRI compatible buffer with ~3ug pSP64-NSERT-poly(A) and 5ul EcoRI.
Incubate 2 hours each, 37C.
Save some plasmid for the gel below.
Pour a 1% agarose gel and run ~100ng of digest and undigested plasmid, to check for cutting.
Assuming you observe complete digestion, clean up reactions using PCR clean up kit, pooling repeats of the same plasmid digest at this stage.
Quantify using nanodrop to have an idea for concentration.
Set up SP6 reaction master mix, from the kit reagents: 1x5xTxn optimized 5x buffer4ul 20ulDTT (100mM)2ul 10ulRecombinant RNasin0.75ul 3.75ul10mM rATP1ul 5ul10mM rUTP1ul 5ul10mM rCTP1ul 5ul10mM rGTP1ul 5ul4tUTP (10mM)2ul 10ulSP6 RNA Polymerase1ul 5ul.
Make new tubes of 6.25ul of linarized plasmid DNA.
For a kit positive control, dilute 1ul of the provided standard with 5.25ul of water.
Concentrations should be in the range of 100-200 ng/ul.
Add 13.75ul of SP6 reaction master mix to each tube of linearized DNA.
Incubate 1-2hr in 30/37C water bath.
Remove 2ul into PCR tubes for later gel.
To the rest, add 1ul of RQ1 RNase-Free DNase.
Incubate 15min at 37C.
To each reaction, add 40ul Ampure XP beads and mix well with pipette.
Let sit RT for 5min.
Collected beads to side in a magnetic rack.
Aspirated supernatant, all.
Add ~500ul 80% etOH onto beads.
Let sit ~30 seconds, then aspirate off.
Add ~500ul 80% etOH onto beads.
Let sit ~30 seconds, then aspirate off.
Make sure to get everything out of the bottom, small tip may help.
Let beads dry in the rack with a kimwipe over the open tubes, 10min RT.
Resuspend beads in 20ul hyclone H_2O.
Run ~3ul on 1% agarose TAE gel for about 20min at 100V with the NEB 1kb ladder.
Visualize with your favorite dye (we use sybrsafe now).
Quantify on qubit, dilute.
0.1ng/ul is a good working mix.
Benjy suggests using 4ng spikein (each) : 100ug total RNA.
Darach's had success with 1ng for ~5e7 cells (for qPCR normalization).