Showing posts with label Graphs. Show all posts
Showing posts with label Graphs. Show all posts

Monday, June 22, 2015

6 22 2015 CARM1 qPCR

Today I ran a qPCR for CARM1 using cDNA created by Sam last week. On friday Steven got the initial run of sequenced samples to check that all populations produced the same sequence. He selected CARM1 as being the most similar with the other primers having one issue or another. I put together a qPCR plate with all the cDNA samples to see how CARM1 differs between populations and treatment/control.

Primer Used:
1642CARM1_FWDTGGTTATCAACAGCCCCGACJH5/21/20152055O.luridaHistone-arginine methyltransferase CARM1 (EC 2.1.1.-) (EC 2.1.1.125) (Coactivator-associated arginine methyltransferase 1) (Protein arginine N-methyltransferase 4)Q6DC04
1641CARM1_REVGTTGTTGACCCCAGGAGGAGJH5/21/20152055O.luridaHistone-arginine methyltransferase CARM1 (EC 2.1.1.-) (EC 2.1.1.125) (Coactivator-associated arginine methyltransferase 1) (Protein arginine N-methyltransferase 4)Q6DC04

qPCR Master Mix Reagent Table:
VolumeReactions X55
Ssofast Evagreen MM 10550
FWD Primer0.527.5
REV Primer0.527.5
Nuclease Free H2O8440
cDNA1
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 19 ul Master Mix to each tube
  5. Pipetted appropriate cDNA sample to each tube
    1. Due to pipette error only 3 NTCs were run
  6. Centrifuged plate at 2000 rpm for 1 minute
  7. Ran Program Below
Sybr New Plate+Sybr cDNA 60 melt 2 Read
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:
1234567
DNased 42215 HC1DNased 42215 NC1DNased 42215 SC1DNased 42215 HT1 1DNased 42215 NT1 1DNased 42215 ST1 1NTC
DNased 42215 HC2DNased 42215 NC2DNased 42215 SC2DNased 42215 HT1 2DNased 42215 NT1 2DNased 42215 ST1 2NTC
DNased 42215 HC3DNased 42215 NC3DNased 42215 SC3DNased 42215 HT1 3DNased 42215 NT1 3DNased 42215 ST1 3NTC
DNased 42215 HC4DNased 42215 NC4DNased 42215 SC4DNased 42215 HT1 4DNased 42215 NT1 4DNased 42215 ST1 4
DNased 42215 HC5DNased 42215 NC5DNased 42215 SC5DNased 42215 HT1 5DNased 42215 NT1 5DNased 42215 ST1 5
DNased 42215 HC6DNased 42215 NC6DNased 42215 SC6DNased 42215 HT1 6DNased 42215 NT1 6DNased 42215 ST1 6
DNased 42215 HC7DNased 42215 NC7DNased 42215 SC7DNased 42215 HT1 7DNased 42215 NT1 7DNased 42215 ST1 7
DNased 42215 HC8DNased 42215 NC8DNased 42215 SC8DNased 42215 HT1 8DNased 42215 NT1 8DNased 42215 ST1 8

Results:

HC1-8 Amplification


 HC1-8 Melt Curve
NC1-8 Amplification
 NC1-8 Melt Curve
SC1-8 Amplification
 SC1-8 Melt Curve
HT1-8 Amplification
 HT1-8 Melt Curve
NT1-8 Amplification
 NT1-8 Melt Curve
ST1-8 Amplification
 ST1-8 Melt Curve
NTC 1-3 Amplification
 NTC 1-3 Melt Curve

As you can see there is good amplification amoung all samples but without running the data through PCR Miner I can't distinguish a difference. Also there was some amplification in the NTC but it appears to be nonspecific with a much higher melting temp than the actual products. These high temp amplifiers don't appear in any of the other samples so I'm not worried about them as possible contamination. I've run a PCR on some more flanking primers to have them sequenced and am running a qPCR for Actin to see if we have similar results.

You can see the raw data file here.
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Thursday, May 28, 2015

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. 

1626HSP70c_FWDAGGAAAGGTCGGGAGAGGAAJH5/21/20152055O.luridaHeat shock 70 kDa protein 12A
1625HSP70c_REVACCTCGGACTTTGGACGAACJH5/21/20152055O.luridaHeat shock 70 kDa protein 12A
1624p29ING4_FWDTACCTTTGGGCTTCACCGTCJH5/21/20152055O.luridaInhibitor of growth protein 4 (p29ING4)
1623p29ING4_REVGTCCATCACACACCCCTCAGJH5/21/20152055O.luridaInhibitor of growth protein 4 (p29ING4)
1622CerS2_FWDTTGTCGGTCTCCTCCTGCTAJH5/21/20152055O.luridaCeramide synthase 2 (CerS2) (LAG1 longevity assurance homolog 2)
1621CerS2_REVCCGTCTTCTGAGCCATCGTTJH5/21/20152055O.luridaCeramide synthase 2 (CerS2) (LAG1 longevity assurance homolog 2)
1620GABABR1_FWDCCGAGGAGGACACGAAACTCJH5/21/20152055O.luridaGamma-aminobutyric acid type B receptor subunit 1 (GABA-B receptor 1) (GABA-B-R1) (GABA-BR1) (GABABR1) (Gb1)
1619GABABR1_REVCGGACAGGTTCTGGATTCCGJH5/21/20152055O.luridaGamma-aminobutyric acid type B receptor subunit 1 (GABA-B receptor 1) (GABA-B-R1) (GABA-BR1) (GABABR1) (Gb1)
1618HSP70d_FWDTTTGTCTCACCGGCTTTGTGJH5/21/20152055O.luridaHeat shock 70 kDa protein 6 (Heat shock 70 kDa protein B')
1617HSP70d_REVGACATGAGACCAAAGACGCCJH5/21/20152055O.luridaHeat shock 70 kDa protein 6 (Heat shock 70 kDa protein B')
1616THRa_FWDGACACTATCCTCACTCGGCGJH5/21/20152055O.luridaThyroid hormone receptor alpha (Nuclear receptor subfamily 1 group A member 1)
1615THRa_REVGGGTGCCGAGTAAACAAGGAJH5/21/20152055O.luridaThyroid hormone receptor alpha (Nuclear receptor subfamily 1 group A member 1)
1614Defensin_FWDTCTAGCGGAGTTTGTTGGGGJH5/21/20152055O.luridaBig defensin
1613Defensin_REVATGGCTGTCGGAGGAGGATTJH5/21/20152055O.luridaBig defensin
1612GRB2_FWDAACTTTGTCCACCCAGACGGJH5/21/20152055O.luridaGrowth factor receptor-bound protein 2 (Adapter protein GRB2) (Protein Ash) (SH2/SH3 adapter GRB2)
1611GRB2_REVCCAGTTGCAGTCCACTTCCTJH5/21/20152055O.luridaGrowth factor receptor-bound protein 2 (Adapter protein GRB2) (Protein Ash) (SH2/SH3 adapter GRB2)
1610H3.3_FWDCACGCTCTCCTCGAATCCTCJH5/21/20152055O.luridaHistone H3.3
1609H3.3_REVAAGTTGCCTTTCCAGCGTCTJH5/21/20152055O.luridaHistone H3.3
1608H2A.V_FWDTGCTTTCTGTGTGCCCTTCTJH5/21/20152055O.luridaHistone H2A.V (H2A.F/Z) (Fragment)
1607H2A.V_REVTATCACACCCCGTCACTTGCJH5/21/20152055O.luridaHistone H2A.V (H2A.F/Z) (Fragment)
1606H2A_FWDGCTGGGGTTTTTCTGGGTCTJH5/21/20152055O.luridaHistone H2A
1605H2A_REVGGAACTACGCCGAGAGAGTGJH5/21/20152055O.luridaHistone H2A
After producing the working stocks I then made a 10 reaction master mix. 

Master Mix reagent table
VolumeReactions X10
Ssofast Evagreen MM10100
FWD Primer0.55
REV Primer0.55
Nanopure H2O880
cDNA1
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:
HSP70cP29INGCerS2GABABRHSP70dTHRaDefensinGRB2H3.3H2A.VH2A
123456789101112
NTCNTCNTCNTCNTCNTCNTCNTCNTCNTCNTC
NT1NT1NT1NT1NT1NT1NT1NT1NT1NT1NT1
HT1HT1HT1HT1HT1HT1HT1HT1HT1HT1HT1
ST1ST1ST1ST1ST1ST1ST1ST1ST1ST1ST1
NC1NC1NC1NC1NC1NC1NC1NC1NC1NC1NC1
HC1HC1HC1HC1HC1HC1HC1HC1HC1HC1HC1
SC1SC1SC1SC1SC1SC1SC1SC1SC1SC1SC1
NTCNTCNTCNTCNTCNTCNTCNTCNTCNTCNTC

qPCR Program:
Sybr New Plate+Sybr cDNA 60 melt 2 Read
StepTemperatureTime
Initiation95 C10 min
Elongation95 C30 sec
55 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

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.

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