Tag Archives: cyclooxygenase

5’/3′ RACE PCRs – COX2 Sequence on 5′ & 3′ RACE Libraries (from 20080619)

Ran PCRs on both the 5′ & 3′ RACE libraries created 20080619 with a new COX2 gene-specifc (GSP) primer designed by Steven (CgPGSRACEsrGSP1; SR ID: 1208). Although this primer was designed to obtain additional 5′ sequence, it was used with both 5′ and 3′ libraries as a precaution in case it accidentally designed on the wrong strand. PCR rxn was set up according to the Clontech SMARTer RACE cDNA Amplification Kit. Master mix calcs are here. PCR cycling followed “Program 1″ from the Clontech manual for 25 cycles.

After PCR completion, 5uL were transferred to a clean tube and saved, in case this PCR didn’t work and a nested PCR would need to be performed. This is according to the Clontech protocol. Samples were run on a 1.2% agarose gel, as instructed in the Clontech manual.

Results:

No bands of any kind in any sample, including the negative controls (gel not shown). Will perform nested PCR on both libraries in hopes of getting bands.

qPCR – C.gigas COX1/COX2 Tissue Distribution

Performed qPCR using pooled cDNA from 20110311. Pooled 2uL from each of the following samples groups: Dg 3hr C, Gill 1hr C, Gill 1hr E, Mantle 3hr C, and Muscle 3hr C. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). Primers sets run were:

EF1_qPCR_5′,3′ (SR IDs: 309, 310)

Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX1_qPCR_R (SR ID: 1191)- Target = COX1

Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX2_454align1_R (SR ID: 1190) – Target = COX2

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Graphs were generated using the BioRad CFX Manager v2.0 software. Expression was normalized to EF1. Also to note, gene efficiency was assumed as 100% by the software since no standard curve was run on the plate. As such, analysis of this data may not be exact.

It’s clear by examining the graphs that the primers being used to differentiate COX1 and COX2 (since they share a common primer: SRID 1192) are differentially expressed. This indicates that the primer sets are indeed amplifying different targets as hoped. This was the primary intention of this qPCR. However, we also now have an idea of tissue distribution of the two genes, as well as their response to V. vulnificus exposre after 1hr. Next step is to perform this qPCR on all the individuals from this experiment as well as the different tissues.

3’RACE – C.gigas 3’RACE for COX2

Used Cg_COX2_3’RACE_short (SR ID: 1197) & Cg_COX2_3’RACE_long (SR ID: 1196) and the Clonetech SMART RACE cDNA Amplification Kit (unknown acquisition date) to attempt to acquire more 3′ sequence of the C.gigas COX2 isoform. Used Gigas 3’RACE cDNA (from 20080610).

Results:

Gel Loading:

Lane 1: Hyperladder 1

Lane 2: empty

Lane 3: Cg_COX2_3’RACE_long

Lane 4: Cg_COX2_3’RACE_long NTC

Lane 5: empty

Lane 6: Cg_COX2_3’RACE_short

Lane 7: Cg_COX2_3’RACE_short NTC

Lane 8: Hyperladder 1

No products produced. This could be due to a large number of factors. The age of the cDNA (from 20080610) is well beyond what the Clontech manual says for storage term (6 months). Additionally, the Clontech polymerase used was nearly 6 years old. The kit (and its components) are of an unknown age and could factor in to the failure of this procedure. Also, the primers that were designed had less than ideal Tm, per the kit’s recommendations.

May need to sequence some previously purified potential COX2 fragments in order to obtain a more useable region of the gene for RACE.

PCR – New C. gigas COX Primers for Sequencing of Isoforms

Used new primers for obtaining bands for additional sequencing of both COX isoforms in C. gigas. Master mix calcs are here. Master mix shorthand (MM##) is described below:

MM07 – Cg_COX_416_F (SR ID: 1193) + Cg_COX1_qPCR_R (SR ID: 1191) Expected band size (if no intron) = ~1540bp

MM08 – Cg_COX_416_F (SR ID: 1193) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~1540bp

MM09 – Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX1_qPCR_R (SR ID: 1191) Expected band size (if no intron) = ~225bp

MM10 – Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~225bp

MM11 – Cg_COX_1519_F (SR ID: 1146) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~275bp

MM12 – Cg_COX_982_F (SR ID: 1151) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~812bp

Results:

Ladder is Hyperladder I from Bioline.

Master mixes are indicated underneath each group by the labels MM##. The order within each MM group (from left to right) is: template, NTC, NTC.

All bands boxed with green were purified using Millipore’s Ultrafree-DA spin columns. Samples were stored @ -20C in “Sam’s Misc. -20C Box”.

MM07 – Fails to produce any bands of any size. Suggests the presence of intron(s) causing the size of the potential amplicon to exceed the capabilities of the polymerase under these cycling conditions.

MM08 – Produces a band of ~400bp which is well below the expected 1540bp (if no introns) size. Due to the faintness of the band, the band was not excised. Will consult with Steven to see if he thinks it worth repeating to produce sufficient product for sequencing.

MM09 – Produce a ~500bp band. The band was excised. This band size is ~275bp larger than the expected size of 225bp. This implies the presence of an intron in this region. This band size differs from that produced by MM10, which suggests that this primer set can be used for qPCR AND distinguish between the COX1 and COX2 isoforms.

MM10 – Produced a ~700bp band. The band was excised. This band size is ~475bp larger than the expected size of 225bp. This implies the presence of an intron in this region. This band size differs from that produced by MM09, which suggests that this primer set can be used for qPCR AND distinguish between the COX1 and COX2 isoforms.

MM11 – Produced multiple bands, of which two were excised; a ~3000bp band and a ~600bp band. These bands were excised solely based on their intensity and their immediate useability for sequencing. Will discuss with Steven on whether or not this should be repeated and the other bands excised for sequencing purposes. Both bands that were excised exceed the expected band size of ~275bp, suggesting the presence of multiple introns. Additionally, the presence of so many products suggests that the primers are not very specific, in regards to their target.

MM12 – An extremely faint band of ~350bp can be seen, however, due to it’s faintness and it’s small size (expected size was ~812bp), the band was not excised. Will discuss with Steven to see if this warrants repeating to accumulate sufficient product for sequencing purposes. No amplification of any larger products suggests the presence of introns, causing the size of the potential amplicon to exceed the capabilities of the polymerase under these cycling conditions. This is also confirmed by the MM11 PCR results in which a 3000bp band was produced. Since the primer set in MM12 has an additional 600bp at the 5′ end, this has already exceeded the abilities of the polymerase, even if this addtional 600bp does NOT include an additional intron. However, it is curious that the MM12 primer set does not produce smaller, spurious PCR products as is seen in the MM11 primer set (these two primer sets both use the same forward primer).

Genomic PCR – Repeat of C.gigas COX genomic PCR from 20110118

This was repeated to generate more PCR product for sequencing purposes. PCR master mix calcs and cycling params are here. Master mixes 04 and 05 (MM04 and MM05) were repeated to gain more PCR product from the faint 550bp & 1500bp bands(MM04) and 5000bp band (MM05).

MM04 – Cg_COX_982_F (SR ID: 1151) + Cg_COX_1545_R (SR ID: 1148) Band size w/o intron = ~550bp

MM05 – Cg_COX_982_F (SR ID: 1151) + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~1130bp

Results:

Gel was run on 20110203

Samples on the left portion of the gel are from the MM04 primer combo and those on the right are from the MM05. Boxed bands were excised, purified using Millipore Ultra DA-free spin columns and stored @ -20C in Sam’s “Misc. -20C Box.”

Interestingly in the MM05 set, inconsistent, faint bands of ~400-500bp are visible. These were not visible the first time this PCR was conducted (see 20110118), but the exposure of the gel image wasn’t turned up as high as in this image. Due to their inconsistency and extremely low yield, these bands were not excised.

Genomic PCR – C.gigas cyclooxygenase (COX) genomic sequence

Attempt to obtain full genomic sequence for C.gigas COX. PCR set up/cycling params/etc are here. Primer set combinations(master mixes) are as follows:

MM01 – Cg_COX_5’UTR_3_F (SR ID: 1150) + Cg_COX_1009_R (SR ID: 1147) Band size w/o intron = ~1000bp

MM02 – “” + Cg_COX_1545_R (SR ID: 1148) Band size w/o intron = ~1540bp

MM03 – “” + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~2135bp

MM04 – Cg_COX_982_F (SR ID: 1151) + Cg_COX_1545_R (SR ID: 1148) Band size w/o intron = ~550bp

MM05 – “” + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~1130bp

MM06 – Cg_COX_1519_F (SR ID: 1146) + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~620bp

Results:

Bioline Hyperladder I used for marker. Gel is loaded with template samples at the far left of each master mix group with two no template controls (NTC) in the remaining two wells of each master mix group. All NTCs on the gel are clean.

All bands surrounded by a green box were excised from the gel.

MM01, MM02 and MM03 – The smallest expected band (i.e. no intron present) would have been 1000bp in MM01. Instead, we see faint banding of multiple sizes less than 1000bp in both MM01 and MM02. MM03 fails to produce any bands. This potentially suggests a couple of things. Firstly, the multiple banding produced in MM01 and MM02 suggests that the PCR conditions lead to some non-specific priming and should be optimized. Secondly, the fact that no bands were produced that are equal to or larger than the “no intron size” suggests that intron(s) may exist in the 5′ region of the COX gene and are large enough that the polymerase had insufficient time/ability to amplify.

MM04 – Three distinct bands were produced: 2000bp, 1500bp and 550bp. The size of band that would have been produced had an intron NOT been present would have been ~550bp. A band of this size was produced in this PCR reaction. However, two additional bands were produced. The presence of these two larger bands lends additional evidence for the existence of multiple isoforms of COX (which is also supported by the fact that multiple isoforms of COX are known to exist in most other species). The 2000bp band was excised and purified with Millipore Ultra-free DA spin columns and stored @ -20C until a sequencing plate is readied.

MM05 – A distinct band of ~5000bp was produced. This is significantly larger than the “no intron size” of ~1130bp, suggesting the presence of an intron. This band was excised, but not purified due to the low concentration of DNA in the gel. The gel slice was stored @ -20C until this PCR reaction could be repeated to accumulate sufficient product for sequencing.

MM06 – A distinct band of ~2200bp was produced. This is significantly larger than the “no intron size” of ~620bp, suggesting the presence of an intron. The band was excised and purified with Millipore Ultra-free DA spin columns and stored @ -20C until a sequencing plate is readied.

The PCR reactions reveal the presence of intron(s) in the COX gene we’re investigating as well as providing evidence for the existence of multiple isoforms in C.gigas. Since the PCR products that have been excised for sequencing are so large, additional primers will need to be designed closer to the introns in order to generate smaller products that can be fully sequenced. Additionally, all reactions using the primer designed to anneal in the 5’UTR of COX (SR ID: 1150) failed to produce useful results. This is either due to poor performance of the primer under these reaction conditions or due to the presence of a large intron in the 5′ region of the gene. Additional reverse primers will be designed that anneal closer to the 5′ portion of the COX gene in hopes of characterizing the 5′ genomic sequence better.

After speaking with Steven today about the potential existence/”discovery” of multiple isoforms, he decided to map the newly-released C.gigas 454 NGS data to the existing COX coding sequence in GenBank (FJ375303). The alignment is shown below.

The two 454 reads that map closest to the 5′ end of the COX coding sequence match up nearly perfectly, with periodic SNPs. The remaining 454 reads that map to the COX coding sequence are very different and provide very good evidence of a previously unidentified isoform of COX in C.gigas. Primers will be designed from both the existing COX sequence in GenBank (FJ375303) and the other potential isoform. These primers will likely be used in both qPCR and for sequencing purposes, in order to be able to distinguish and characterize both isoforms. Additionally, BLASTing will be performed with the sequences from both isoforms to evaluate how they match up with existing COX isoforms in other species.

qPCR – COX qPCR Vibrio Exposure Response Check

Used COX primers (SR IDs 1060, 1061) and cDNA from 20080327, which consisted of 7 control gigas gill and 7 vibrio-exposed (24hrs) gigas gill samples, labeled as C# and VE#, respectively. The experiment was a 24hr. exposure live Vibrio vulnificus, parahaemolyticus Cf = 2.055×10^11 (6.85×10^7 Vibrio cells/oyster).
Note: Used a free sample of 2x Brilliant III Ultra Fast SYBR Green QPCR Master Mix (Stratagene) for this qPCR. Mixed components and set up cycling params according to the manufacturer’s recommendation for the BioRad CFX96.

Master mix calcs are here. Plate layout, cycling params, etc. can be see in the qPCR Report (see Results).

Results:

qPCR Report (PDF).

PCR Miner analysis is here. There appears to be an increase in COX expression in samples exposed to Vibrio sp. (see graph below), however, I have not determined if the results are significant.

qPCR – COX qPCR Primer Test and Tissue Distribution

Used new cyclooxygenase primers (SR IDs 1060, 1061) to see how they performed and to evaluate tissue distribution. Tissue distribution was evaluated using the following cDNAs made on 10/27/10 from Emma:

Gigas Digestive Gland

Gigas Gill

Gigas Mantle

Gigas Muscle

qPCR Master Mix calcs are here. Plate layout, cycling parameters, etc can be found in the qPCR Report (see Results).

Results:

qPCR Report (PDF).

Amplification is present in all four tissue types and the melting curve looks good. So, these primers are good to go. Steven suggests checking to see if we see a change in gene expression from an old experiment of Gigas exposed to high levels of Vibrio tubiashii. Will round up some old cDNA for this.