5 27 2015 New Primer Check (1-12)

Yesterday I received the new primers that I designed using the oly transcriptome. You can read about that here. Sam rehydrated the original stocks and I produced work stocks from them to use in a plate.

To make the working stock of the primers.

1. 90 ul of Nuclease free (or Nanopure) H20 to 1.5 ml tube
2. 10 ul of Stock primers
3. Vortex briefly

I made these carefully in order to produce the following 12 pairs of primers. 

Hspb11_FWD	ATGTTTCCTGGTCTCCGTCA	JH	5/21/2015	20	55		O.lurida	Heat shock protein beta-11 (Hspb11) (Placental protein 25) (PP25)
Hspb11_REV	CATCAACGCCAGGGGAACTT	JH	5/21/2015	20	55		O.lurida	Heat shock protein beta-11 (Hspb11) (Placental protein 25) (PP25)
GDF-8_FWD	CCGTGGATGTCGCAGAAAGA	JH	5/21/2015	20	55		O.lurida	Growth/differentiation factor 8 (GDF-8) (Myostatin) (Myostatin-1) (zfMSTN-1) (Myostatin-B)
GDF-8_REV	CTGCTTTCTCCGTCCCCTTT	JH	5/21/2015	20	55		O.lurida	Growth/differentiation factor 8 (GDF-8) (Myostatin) (Myostatin-1) (zfMSTN-1) (Myostatin-B)
HSP70b_FWD	AAGTACCTTGGGGAGCTTGC	JH	5/21/2015	20	55		O.lurida	Heat shock 70 kDa protein 12B
HSP70b_REV	TCCACAGACTTTCCTCCCCA	JH	5/21/2015	20	55		O.lurida	Heat shock 70 kDa protein 12B
GRP-78_FWD	GAGAAACCACGCAGGGAGAA	JH	5/21/2015	20	55		O.lurida	78 kDa glucose-regulated protein (GRP-78) (Heat shock 70 kDa protein 5) (Immunoglobulin heavy chain-binding protein) (BiP)
GRP-78_REV	CATCAGCATCGAAGGCAACG	JH	5/21/2015	20	55		O.lurida	78 kDa glucose-regulated protein (GRP-78) (Heat shock 70 kDa protein 5) (Immunoglobulin heavy chain-binding protein) (BiP)
CARM1_FWD	TGGTTATCAACAGCCCCGAC	JH	5/21/2015	20	55		O.lurida	Histone-arginine methyltransferase CARM1 (EC 2.1.1.-) (EC 2.1.1.125) (Coactivator-associated arginine methyltransferase 1) (Protein arginine N-methyltransferase 4)
CARM1_REV	GTTGTTGACCCCAGGAGGAG	JH	5/21/2015	20	55		O.lurida	Histone-arginine methyltransferase CARM1 (EC 2.1.1.-) (EC 2.1.1.125) (Coactivator-associated arginine methyltransferase 1) (Protein arginine N-methyltransferase 4)
BMP-2_FWD	TGAAGGAACGACCAAAGCCA	JH	5/21/2015	20	55		O.lurida	Bone morphogenetic protein 2 (BMP-2) (Bone morphogenetic protein 2A) (BMP-2A)
BMP-2_REV	TCCGGTTGAAGAACCTCGTG	JH	5/21/2015	20	55		O.lurida	Bone morphogenetic protein 2 (BMP-2) (Bone morphogenetic protein 2A) (BMP-2A)
PGE/EP4_FWD	ACAGCGACGGACGATTTTCT	JH	5/21/2015	20	55		O.lurida	Prostaglandin E2 receptor EP4 subtype (PGE receptor EP4 subtype) (PGE2 receptor EP4 subtype) (Prostanoid EP4 receptor)
PGE/EP4_REV	ATGGCAGACGTTACCCAACA	JH	5/21/2015	20	55		O.lurida	Prostaglandin E2 receptor EP4 subtype (PGE receptor EP4 subtype) (PGE2 receptor EP4 subtype) (Prostanoid EP4 receptor)
CRAF1_FWD	AGCAGGGCATCAAACTCTCC	JH	5/21/2015	20	55		O.lurida	TNF receptor-associated factor 3 (EC 6.3.2.-) (CD40 receptor-associated factor 1) (CRAF1) (TRAFAMN)
CRAF1_REV	ACAAGTCGCACTGGCTACAA	JH	5/21/2015	20	55		O.lurida	TNF receptor-associated factor 3 (EC 6.3.2.-) (CD40 receptor-associated factor 1) (CRAF1) (TRAFAMN)
NFKBina_FWD	GATGGCGGTGCATGTGTTAG	JH	5/21/2015	20	55		O.lurida	NF-kappa-B inhibitor alpha (I-kappa-B-alpha) (IkB-alpha) (IkappaBalpha) (REL-associated protein pp40)
NFKBina_REV	CGAGGAGAACCTTGTGCAGT	JH	5/21/2015	20	55		O.lurida	NF-kappa-B inhibitor alpha (I-kappa-B-alpha) (IkB-alpha) (IkappaBalpha) (REL-associated protein pp40)
PGRP-S_FWD	GAGACTTCACCTCGCACCAA	JH	5/21/2015	20	55		O.lurida	Peptidoglycan recognition protein 1 (Peptidoglycan recognition protein short) (PGRP-S)
PGRP-S_REV	AACTGGTTTGCCCGACATCA	JH	5/21/2015	20	55		O.lurida	Peptidoglycan recognition protein 1 (Peptidoglycan recognition protein short) (PGRP-S)
TLR2.1_FWD	ACAAAGATTCCACCCGGCAA	JH	5/21/2015	20	55		O.lurida	Toll-like receptor 2 type-1
TLR2.1_REV	ACACCAACGACAGGAAGTGG	JH	5/21/2015	20	55		O.lurida	Toll-like receptor 2 type-1
GDF-8b_FWD	AACTGATTCTGCTCGTCGCA	JH	5/21/2015	20	55		O.lurida	Growth/differentiation factor 8 (GDF-8) (Myostatin)
GDF-8b_REV	TGTTCTTCCACCCACCACTG	JH	5/21/2015	20	55		O.lurida	Growth/differentiation factor 8 (GDF-8) (Myostatin)

After producing the working stocks I then made a 10 reaction master mix. 

Master Mix reagent table

	Volume	Reactions X10
Ssofast Evagreen MM	10	100
FWD Primer	0.5	5
REV Primer	0.5	5
Nanopure H2O	8	80
cDNA	1

Protocol:

1. Added Ssofast to each of 12 tubes
2. Added Nanopure water to each of 12 tubes
3. Carefully added FWD primer, then Reverse primer to the appropriate tube
4. Vortex briefly
5. One Master Mix used per column in plate
6. Pipette 19 ul of master mix to each well for the appropriate column
7. Either No Template Control (NTC) or sample for each Row in the plate
8. 1 ul Sample/NTC per well in the appropriate row

Table Layout:

Hspb11	GDF-8	HSP70b	GRF-78	CARM1	BMP2	PGE/EP4	CRAF1	NFKBina	PGRP-S	TLR2.1	GDF-8b
1	2	3	4	5	6	7	8	9	10	11	12
NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC
NT1	NT1	NT1	NT1	NT1	NT1	NT1	NT1	NT1	NT1	NT1	NT1
HT1	HT1	HT1	HT1	HT1	HT1	HT1	HT1	HT1	HT1	HT1	HT1
ST1	ST1	ST1	ST1	ST1	ST1	ST1	ST1	ST1	ST1	ST1	ST1
NC1	NC1	NC1	NC1	NC1	NC1	NC1	NC1	NC1	NC1	NC1	NC1
HC1	HC1	HC1	HC1	HC1	HC1	HC1	HC1	HC1	HC1	HC1	HC1
SC1	SC1	SC1	SC1	SC1	SC1	SC1	SC1	SC1	SC1	SC1	SC1
NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC

qPCR Program:

Sybr New Plate+Sybr cDNA 60 melt 2 Read
Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	30 sec
	55 C	1 min
Read
	72 C	30 sec
Read
Repeat Elongation 39 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	55 - 95 C	30 sec
	21 C	10 min
End

Results:

HSPb11 Amplification

HSPb11 Melt Curve

GDF-8 Amplification

 GDF-8 Melt Curve

HSP70b Amplification

 HSP70b Melt Curve

GRF-78 Amplification

GRF-78 Melt Curve

CARM1 Amplification

 CARM1 Melt Curve

BMP2 Amplification

BMP2 Melt Curve

PGE/EP4 Amplification

PGE/EP4 Melt Curve

CRAF1 Amplification

CRAF1 Melt Curve

NFKBina Amplification

 NFKBina Melt Curve

PGRP-S Amplification

 PGRP-S Melt Curve

TLR2.1 Amplification

 TLR2.1 Melt Curve

GDF-8b Amplification

 GDF-8b Melt Curve

There was some very interesting results. 

CARM1, BMP2, HSP11b, and PGE/EP4 All showed down regulation from heat shock in all populations.

GDF-8 Had High expression in Fidalgo

CRAF1 had Up regulation in the Oyster Bay population and down regulation in others

PGRP-S had Up regulation in the Dabob population and down regulation in the others

TLR2.1 Had the most interesting response. It was down regulated in the Fidalgo bay population, Equal Expression in the Oyster Bay population, and did not amplify in either Dabob sample. 

HSP70b, GRF-78, NFKBina and GDF-8b had severe issues with the melt curve and odd expression in the samples. These four primers are probably not useful. 

I'm testing the next batch of primers now and will report the results when the come in. 

You can see the raw qPCR file here.