Tuesday, August 11, 2015

8 10 2015 PGEEP4 Mech Stress 2 rep qPCR

Today I ran mechanical stress samples in replicates of two to generate data about expression values. Here I ran PGEEP4 . I ran NTCs on a separate plate using the same reaction volume and 9 ul of PCR water.

Primers:
1638PGE/EP4_FWDACAGCGACGGACGATTTTCTJH5/21/20152055O.luridaProstaglandin E2 receptor EP4 subtype (PGE receptor EP4 subtype) (PGE2 receptor EP4 subtype) (Prostanoid EP4 receptor)P32240
1637PGE/EP4_REVATGGCAGACGTTACCCAACAJH5/21/20152055O.luridaProstaglandin E2 receptor EP4 subtype (PGE receptor EP4 subtype) (PGE2 receptor EP4 subtype) (Prostanoid EP4 receptor)P32240
Reagent Table:

VolumeReactions X58
Ssofast Evagreen MM10580
FWD Primer0.529
REV Primer0.529
1:9 cDNA9
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 11 ul Master Mix to each tube using a  multichannel pipetter
  5. Pipetted 9 ul of 1:9 cDNA each column using a channel pipetter
  6. Centrifuged plate at 2000 rpm for 1 minute
  7. Ran Program Below
Program:
StepTemperatureTime
Initiation95 C10 min
Elongation95 C30 sec
60 C1 min
Read
72 C30 sec
Read
Repeat Elongation 39 times
Termination95 C1 min
55 C1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C55 - 95 C30 sec
21 C10 min
End

Plate Layout:
Rep 1Rep 1Rep 1Rep 2Rep 2Rep 2
789101112
DNased 42215 HM1 1DNased 42215 NM1 1DNased 42215 SM1 1DNased 42215 HM1 1DNased 42215 NM1 1DNased 42215 SM1 1
DNased 42215 HM1 2DNased 42215 NM1 2DNased 42215 SM1 2DNased 42215 HM1 2DNased 42215 NM1 2DNased 42215 SM1 2
DNased 42215 HM1 3DNased 42215 NM1 3DNased 42215 SM1 3DNased 42215 HM1 3DNased 42215 NM1 3DNased 42215 SM1 3
DNased 42215 HM1 4DNased 42215 NM1 4DNased 42215 SM1 4DNased 42215 HM1 4DNased 42215 NM1 4DNased 42215 SM1 4
DNased 42215 HM1 5DNased 42215 NM1 5DNased 42215 SM1 5DNased 42215 HM1 5DNased 42215 NM1 5DNased 42215 SM1 5
DNased 42215 HM1 6DNased 42215 NM1 6DNased 42215 SM1 6DNased 42215 HM1 6DNased 42215 NM1 6DNased 42215 SM1 6
DNased 42215 HM1 7DNased 42215 NM1 7DNased 42215 SM1 7DNased 42215 HM1 7DNased 42215 NM1 7DNased 42215 SM1 7
DNased 42215 HM1 8DNased 42215 NM1 8DNased 42215 SM1 8DNased 42215 HM1 8DNased 42215 NM1 8DNased 42215 SM1 8
All
Amplification
Melt Curve
NTC
Amplification
Melt Curve

The amplification and melt curves look good. There's nothing in the NTCs. I added the Ct values provided by the opticon to a spreadsheet to compare replications. You can see the difference in replicates below.

Rep DiffWell / Set
0.55H_M_1
2.16N_M_1
1.36S_M_1
1.03H_M_2
1.92N_M_2
1.27S_M_2
1.04H_M_3
1.86N_M_3
1.47S_M_3
1.86H_M_4
0.9N_M_4
1.37S_M_4
1.06H_M_5
1.26N_M_5
1.19S_M_5
2.43H_M_6
1.09N_M_6
1.03S_M_6
1.42H_M_7
1.38N_M_7
0.85S_M_7
2.65H_M_8
0.94N_M_8
0.61S_M_8

There's 19 of 24 samples that need to be re run. This trial should be completely re run to improve replicates. 

You can see the raw sample data here and NTCs here.
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