# A few outliers remaining

Integrating last bit of qPCR data into master datasheet. This includes 5 runs post 8/15.

likely error above – second EF1 was 18s test.

CARM
Looks nice, corrected…

Elong factor
Correction is fine, but mechanical reps have some issues

As noticed in last batch of analysis these need to be checked at raw data level

28s

Thoughts on normalizing gene…
Noting that one EF1 rep was thrown out for mechanical (see above). Here is a crude look at EF1, actin, and 28s respectively…

# Finishing out with the mechanical

Currently there is a pretty robust spreadsheet and over the past few days Jake has cranked through some reps to see how the oysters that were mechanically stressed hold up. Below is how these data are integrated.

Currently the 8-10 samples (yellow) have been skipped, but we might have a look.

First up is having a look at the new HSP 70 reps. The mechanical data still needs some better resolution. Hopefully teh 8-10 samples migh shed some light.

Next up is two more reps of PGEEP4.
Looks good, and given the doubling of reps we could easily drop ‘outlier’ runs and still have triplicates, tight triplicates.

GRB2… now good to go, with the first pair of reps dead on.

BMP2…. could use some help from the other mechanical stress runs

TLR….seemed like a relatively easy fix (besides no detection) in that just needed to correct for machine.

And the correction indicating the fact that expression was so low, only able to be detected by Opticon

The 8-15 runs had minimal control and temp samples with mechanical run in dups.

This needs a little carressing before integrating into data.
This should be in two columns with empty cells where no samples were run- in this order.

H_C_1
H_C_2
H_C_3
H_C_4
H_C_5
H_C_6
H_C_7
H_C_8
N_C_1
N_C_2
N_C_3
N_C_4
N_C_5
N_C_6
N_C_7
N_C_8
S_C_1
S_C_2
S_C_3
S_C_4
S_C_5
S_C_6
S_C_7
S_C_8
H_T_1
H_T_2
H_T_3
H_T_4
H_T_5
H_T_6
H_T_7
H_T_8
N_T_1
N_T_2
N_T_3
N_T_4
N_T_5
N_T_6
N_T_7
N_T_8
S_T_1
S_T_2
S_T_3
S_T_4
S_T_5
S_T_6
S_T_7
S_T_8
H_M_1
H_M_2
H_M_3
H_M_4
H_M_5
H_M_6
H_M_7
H_M_8
N_M_1
N_M_2
N_M_3
N_M_4
N_M_5
N_M_6
N_M_7
N_M_8
S_M_1
S_M_2
S_M_3
S_M_4
S_M_5
S_M_6
S_M_7
S_M_8


8-15 run update

Actin

Mechanical looks decent after correcting.

However taken together, bothersome the difference in crude expression levels.

Carm

H2AV
Assuming correction is correct- still a big differences in mechanincal here- could be real.

PGRP
No correction required as these were run on cfx, downside is some reps are not detected that would have been picked up with Opticon.

Do not see be shift in expression of mechanical stressed.

CRAF
Easy correction but skeptical of some very, very low Cts

# Dirty and crude with Oly qPCR

Taking the most decent Ct values, I did the simple and crude calculation, normalizing with EF1 and looking at fold over minimum.

Seems to be EF1 data is skewing. Will take a look with actin and also compare delta Ct, using this as a sound reference.