RT-PCR Process- Teegarden 1/31/2011
1. Making RT working mix:
-Get ice and thaw RNA diluted samples from -80, under hood
-Thaw RT working mix reagents (-20 white freezer-clear box, large caps) on ice, under hood
-Label new set of corresponding 0.5 mL tubes: cDNA, Exp#, initials, date, treatment, cell line
+label one 0.5mL tube for RT Working mix
-Pipette calculated amounts of RT working mix (14x for 12 samples) into ‘RT mix’ tube
+Use filter tips (100µl pipette only for quantities >20µl)
+Vortex
+Keep on ice
-Pipette 5.25µl of RT working mix into each new 0.5mL tube
-Pipette 4.75µl of each corresponding RNA dilutions into the new 0.5mL tubes
+Vortex
+Centrifuge in microcentrifuge @15K at room temperature for 5 mins
2. Running RT Mix:
-Sign off on RT machine (Fleet lab, upstairs, new labs, right side counter) for 2 hours
-Use tray top that has correct size for 0.5mL tubes (may need to change top)
-Place all samples in middle of tray
-Close lid
-Set machine for 1hour 10mins to run with heat
3. Make cDNA pool:
-Retrieve samples from machine and bring under hood
-Pipette 90µl of autoclaved ddH2O to each 0.5mL tube, vortex
-Label one 0.5mL tube for ‘cDNA Pool’
-Add 2µl of each 0.5mL sample into ‘cDNA Pool’ tube, vortex
-Store at -80 in OWN box until ready to do Standard Curve testing (sign up for in G53-buhman lab)
4. Make PCR working mix:
-Thaw PCR working mix reagents (4oclear, upper right, box; -20o2nd shelf, PCR box) on ice, under hood
-See working mix sheet for calculations on how much working mix to make
-Label 1.5mL tube ‘PCR working mix’
-Pipette using filter tips, appropriate amounts of Strategene master mix, Forward primer (18S-standard),
Reverse primer (18S-standard), ROX, and ddH20
+Dilute ROX 1:500 in a 0.5mL tube (499µl ddH2O + 1µlROX), vortex
+Vortex, keep on ice
5. PCR Standard Curve Test:
-Get ice and thaw cDNA pool, under hood
-Label 0.5mL tubes for 1:5 dilutions1:5; 1:25; 1:125; 1:625; 1:3125
+Pipette 20µl of ddH20 into each dilution tube
+Pipette 5µl of cDNA from the full strength pool into the 1:5 tube using filter tips, vortex
+Pipette 5µl of the 1:5 cDNA solution into the 1:25 tube, vortex…etc. for all remaining tubes
-Pipette 20µl of PCR working mix into 21 (triplicate dilutions+standards) 0.2mL tubes, using filter tips
-Pipette 5µl of cDNA as appropriate (triplicate each dilution, 1 full strength cDNA, 1 ddH2O), using filter
tips
-DO NOT LABEL LIDS OF TUBES
+vortex in tray
-Take to Buhman lab (G53) to run test
+Centrifuge using one next to PCR machine until NO bubbles
+Open both sets of doors, place samples vertically into holes
+Open computer program, set samples/protocol
-Run PCR machine for 3 hours (let lamp warm up first)
-Read/save results via print/flashdrive
+Need R2 > 97% Eff.
-Store samples at -80 in OWN box
6. PCR mRNA Test:
-Done once standard curve test is statistically suitable
-Follow same process from step 4
+Use appropriate primers..i.e., testing for VHL=use VHL forward/reverse primers
+Always must have an 18S primer to go with any test to use as a base
-Get ice and thaw mRNA samples on ice while making PCR working mix
-Follow step 5 after dilutions step
+Triplicate using the mRNA samples (NV1-TD3) instead of dilutions
+Follow rest of step 5 protocol
-Store samples at -80 in OWN box when complete
6. PCR Quantifications