qPCR

From trypsinized embryo, resuspend in 50ul PBS
Use 15ul and add 500ul Trizol
Store at -80C

RNA extraction
RNaseZAP everything
From cells in Trizol, incubate RT 5min to permit complete dissociation of nucleoprotein complexes
Add 100ul chloroform
Shake vigorously by hand 15s and incubate at RT 2-3min
Spin max speed 15min 4C
Transfer top aqueous phase to fresh eppies (~200ul)
Precipitate RNA from aqueous phase with 250ul isopropanol
Incubate RT for 10min
Spin max speed 10min 4C
Remove supernatant
Wash RNA pellet once with 75% EtOH (DEPC) 750ul
Spin max speed 5min 4C
Remove EtOH, briefly dry RNA pellet
Dissolve in DEPC H2O 20ul. Pipette up and down a few times to completely dissolve
Incubate at 55-60C 10min to dissolve

DNase treatment (25ul)
10x Turbo DNase buffer 2.5
Turbo DNase 1
RNA 20
DEPC H2O 1.5

Incubate 37C 30min
Add 0.1 volume DNase Inactivation Reagent (resuspend before adding) (2.5ul)
Mix well
Incubate 5min RT. Mix 2-3 times
Spin 10000g for 1.5min and transfer RNA to new tube

cDNA synthesis (use PCR machines for incubation)
RNA (up to 5ug) 8
50ng/ul random hexamers 1
10mM dNTP mix 1
Total 10ul

Incubate 65C 5min
Place on ice for at least 1 min

10x RT buffer 2
25mM MgCl2 4
0.1M DTT 2
RNaseOUT (40U/ul) 1
SuperScript III RT (200U/ul) 1
Total 10ul

Add 10ul of cDNA synthesis mix to each RNA/ primer mixture
Mix gently and spin down
10min 25C
50min 50C
5 min 85C
Chill on ice

Add 1ul of RNase H to each tube to remove RNA template
Incubate 20min 37C

QPCR
SYBR Green qPCR Supermix 25
5uM F primer 3.6
5uM R primer 3.6
cDNA 2
DEPC H2O 15.8
Total 50ul

50C 2min
95C 10min
95C 15s
60C 60s
Repeat Steps 3 and 4 39x
Melting curve