5 28 2015 New Primer Check (13-23)

Today I completed the primer checks for the new primers. This run is less promising than yesterdays as it doesn't have any primers that show any real difference in them. There were more than a couple that failed in some manner.

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. 

1626	HSP70c_FWD	AGGAAAGGTCGGGAGAGGAA	JH	5/21/2015	20	55		O.lurida	Heat shock 70 kDa protein 12A
1625	HSP70c_REV	ACCTCGGACTTTGGACGAAC	JH	5/21/2015	20	55		O.lurida	Heat shock 70 kDa protein 12A
1624	p29ING4_FWD	TACCTTTGGGCTTCACCGTC	JH	5/21/2015	20	55		O.lurida	Inhibitor of growth protein 4 (p29ING4)
1623	p29ING4_REV	GTCCATCACACACCCCTCAG	JH	5/21/2015	20	55		O.lurida	Inhibitor of growth protein 4 (p29ING4)
1622	CerS2_FWD	TTGTCGGTCTCCTCCTGCTA	JH	5/21/2015	20	55		O.lurida	Ceramide synthase 2 (CerS2) (LAG1 longevity assurance homolog 2)
1621	CerS2_REV	CCGTCTTCTGAGCCATCGTT	JH	5/21/2015	20	55		O.lurida	Ceramide synthase 2 (CerS2) (LAG1 longevity assurance homolog 2)
1620	GABABR1_FWD	CCGAGGAGGACACGAAACTC	JH	5/21/2015	20	55		O.lurida	Gamma-aminobutyric acid type B receptor subunit 1 (GABA-B receptor 1) (GABA-B-R1) (GABA-BR1) (GABABR1) (Gb1)
1619	GABABR1_REV	CGGACAGGTTCTGGATTCCG	JH	5/21/2015	20	55		O.lurida	Gamma-aminobutyric acid type B receptor subunit 1 (GABA-B receptor 1) (GABA-B-R1) (GABA-BR1) (GABABR1) (Gb1)
1618	HSP70d_FWD	TTTGTCTCACCGGCTTTGTG	JH	5/21/2015	20	55		O.lurida	Heat shock 70 kDa protein 6 (Heat shock 70 kDa protein B')
1617	HSP70d_REV	GACATGAGACCAAAGACGCC	JH	5/21/2015	20	55		O.lurida	Heat shock 70 kDa protein 6 (Heat shock 70 kDa protein B')
1616	THRa_FWD	GACACTATCCTCACTCGGCG	JH	5/21/2015	20	55		O.lurida	Thyroid hormone receptor alpha (Nuclear receptor subfamily 1 group A member 1)
1615	THRa_REV	GGGTGCCGAGTAAACAAGGA	JH	5/21/2015	20	55		O.lurida	Thyroid hormone receptor alpha (Nuclear receptor subfamily 1 group A member 1)
1614	Defensin_FWD	TCTAGCGGAGTTTGTTGGGG	JH	5/21/2015	20	55		O.lurida	Big defensin
1613	Defensin_REV	ATGGCTGTCGGAGGAGGATT	JH	5/21/2015	20	55		O.lurida	Big defensin
1612	GRB2_FWD	AACTTTGTCCACCCAGACGG	JH	5/21/2015	20	55		O.lurida	Growth factor receptor-bound protein 2 (Adapter protein GRB2) (Protein Ash) (SH2/SH3 adapter GRB2)
1611	GRB2_REV	CCAGTTGCAGTCCACTTCCT	JH	5/21/2015	20	55		O.lurida	Growth factor receptor-bound protein 2 (Adapter protein GRB2) (Protein Ash) (SH2/SH3 adapter GRB2)
1610	H3.3_FWD	CACGCTCTCCTCGAATCCTC	JH	5/21/2015	20	55		O.lurida	Histone H3.3
1609	H3.3_REV	AAGTTGCCTTTCCAGCGTCT	JH	5/21/2015	20	55		O.lurida	Histone H3.3
1608	H2A.V_FWD	TGCTTTCTGTGTGCCCTTCT	JH	5/21/2015	20	55		O.lurida	Histone H2A.V (H2A.F/Z) (Fragment)
1607	H2A.V_REV	TATCACACCCCGTCACTTGC	JH	5/21/2015	20	55		O.lurida	Histone H2A.V (H2A.F/Z) (Fragment)
1606	H2A_FWD	GCTGGGGTTTTTCTGGGTCT	JH	5/21/2015	20	55		O.lurida	Histone H2A
1605	H2A_REV	GGAACTACGCCGAGAGAGTG	JH	5/21/2015	20	55		O.lurida	Histone H2A

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:

HSP70c	P29ING	CerS2	GABABR	HSP70d	THRa	Defensin	GRB2	H3.3	H2A.V	H2A
1	2	3	4	5	6	7	8	9	10	11	12
NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC	NTC
NT1	NT1	NT1	NT1	NT1	NT1	NT1	NT1	NT1	NT1	NT1
HT1	HT1	HT1	HT1	HT1	HT1	HT1	HT1	HT1	HT1	HT1
ST1	ST1	ST1	ST1	ST1	ST1	ST1	ST1	ST1	ST1	ST1
NC1	NC1	NC1	NC1	NC1	NC1	NC1	NC1	NC1	NC1	NC1
HC1	HC1	HC1	HC1	HC1	HC1	HC1	HC1	HC1	HC1	HC1
SC1	SC1	SC1	SC1	SC1	SC1	SC1	SC1	SC1	SC1	SC1
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:

HSP70c Amplification

 HSP70c Melt Curve

p29ING Amplification

 p29ING Melt Curve

CerS2 Amplification

 CerS2 Melt Curve

GABABR Amplification

 GABABR Melt Curve

HSP70d Amplification

 HSP70d Melt Curve

THRa Amplification

 THRa Melt Curve

Defensin Amplification

 Defensin Melt Curve

GRB2 Amplification

 GRB2 Melt Curve

H3.3 Amplification

 H3.3 Melt Curve

H2A.V Amplification

 H2A.V Melt Curve

H2A Amplification

 H2A Melt Curve

Posted below are assumptions based on the very limited data from this check. It is only being produced to eliminate useless primers from future tests and highlight possibly interesting primers. Only with further testing will we be able to determine true trends. 

CerS2, HSP70d, THRa, and Defensin failed due to no amplification or poor melt curve conditions. 

HSP70c produced a weird bump inline with the other melt curves. This primer should probably not be used due to the inability to eliminate this issue.

H3.3, H2A.V, and H2A all showed similar results with H2A being very similar in all samples. These could be candidates for normalizing genes to use with Actin. 

p29ING also appeared to have uniform expression in all samples. This could also be another normalizing gene.

GABABR appeared to have down regulation in Heat Stress for Fidalgo and Dabob but there's no way to tell unless we run more replicates. 

GRB2 had some variable expression between heat shock and control samples but we'll need to run further tests to ensure this. 

Tomorrow I should finalize a list of the primers of interest.  Hopefully next week I'll be able to run them with a full complement of samples and normalizing genes. 

You can find the raw qPCR data here.