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RT-PCR primer design


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#1 Jevandrix

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Posted 30 May 2011 - 10:24 PM

Hi,

I need a huge help from all the experts here, since I am new in primer designing. My experiment is on evaluating the expression level of known immune related gene in fish challenged with pathogens. The followings are the list my question:

1. I have obtained the source sequence of many immune-genes from NCBI. The title of the genes varies with partial cd, complete cd, mRNA and sometimes only gene. What does this signifies? In case of expression study, do we have to design primer based on cd seq or is it fine if we use the whole gene seq?

2. What is RefSeq? Why it's important? Is it actually the species of interest's gDNA or model organism's gDNA? What if reference sequence is unavailable?

3. What is the role of BLAST in primer designing? Why alignment is necessary? From my tiny understanding, is it not we just search the gene of interest's sequence and pick primers out of it?

4. Is it fine to design primers through NCBI's pick primer feature or is it better to use software such as Primer 3 or PerlPrimer?

5. To evaluate a primer designed based on cDNA sequence, does it make sense if I evaluate the primer on gDNA and run it on gel or do I have to test it on cDNA?

6. In the case of designing a primer for a non-model organism, it is suggested that we use the sequence of the gene of interest sequenced from a different organism as a base. Does it mean I just find the gene's sequence in NCBI> narrow the genera or species range to the most closely related > Pick primers based on the sequence > test it on the organism of interest?

As you all have notice there a lot of potential dumb questions there. So, major assistance from all of you is serious needed. Thank you in advance.

-Jevandrix86

#2 Jevandrix

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Posted 30 May 2011 - 10:43 PM

Another question; Is it recommended to design primer for RT-PCR to span exon-exon junction to minimise gDNA amplification. Does this only applicable if the gDNA of the organism of interest is available?

#3 Trof

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Posted 31 May 2011 - 12:36 AM

1. I have obtained the source sequence of many immune-genes from NCBI. The title of the genes varies with partial cd, complete cd, mRNA and sometimes only gene. What does this signifies? In case of expression study, do we have to design primer based on cd seq or is it fine if we use the whole gene seq?

"cds" means coding sequence, that usually means mRNA not DNA sequence. Complete cds should contain whole reading frame, partial only a part. mRNA should be complete mRNA molecule, that means coding sequence and 3' and 5' untranslated region. The "gene" however is more likely to contain genomic DNA sequence.

2. What is RefSeq? Why it's important? Is it actually the species of interest's gDNA or model organism's gDNA? What if reference sequence is unavailable?

RefSeq is a reference sequence, that should be validated and used for all subsequent applications. Every species has it's own reference sequence. Uncommon organisms however may not have a RefSeq, then you have to search other nucleotide sequences. Best way do get RefSeq, if there is one, is to search the NCBI modul Gene for your gene of interest and your species.

3. What is the role of BLAST in primer designing? Why alignment is necessary? From my tiny understanding, is it not we just search the gene of interest's sequence and pick primers out of it?

Some sequences may be repetitive in genome or the gene of interest has a pseudogene or a duplicate. You need to check that they bind specifically only your gene and not something else. You can either BLAST each of put them in the PrimerBLAST (you need to add amplicon sequence there too) that will tell you all possible nonspecific products.

4. Is it fine to design primers through NCBI's pick primer feature or is it better to use software such as Primer 3 or PerlPrimer?

PrimerBLAST uses primer3 algorithm for primer design (I don't know PearlPrimer). PrimerBLAST is nice for finding specific primers, because it already BLASTs the potential primers and selects specific one. It can also automatically design intron spanning primers, which are recomended for mRNA quantification. On the other hand it's not possible to mark taget and excluded regions, as does primer3 which is sometimes important if you want to design primers in a specific exon or something like that. Also since primer3 doesn't BLAST primers (it only allows you to mask out repetitive sequences) you need to do it yourself.

5. To evaluate a primer designed based on cDNA sequence, does it make sense if I evaluate the primer on gDNA and run it on gel or do I have to test it on cDNA?

Usually not. The intron spanning primers are recomended for qPCR and ideally they should not amplify gDNA at all.

Is it recommended to design primer for RT-PCR to span exon-exon junction to minimise gDNA amplification. Does this only applicable if the gDNA of the organism of interest is available?

Well if you don't know where are your exons you can hardly design primers on exon-exon junctions. It the case you don't have such primers you should treat your samples with DNase and run RT- control everytime.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

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#4 Jevandrix

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Posted 01 June 2011 - 01:31 AM

Dear Mr. Trof,

Thank you a lot, the PhD student working together with me is not willing to share her knowledge...minimum guidance. I hope to get more guidance from you. God bless you.

Much obliged,
Jevandrix

#5 Jevandrix

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Posted 05 June 2011 - 08:36 PM

Hi Miss/Mrs. Trof & everyone,

I hope you could clarify one thing regarding the BLAST in primer designing. The questions are:

1. When we BLAST for the specificity of the primer, does it have to be species-specific or not. For example if I have primers for gene X in Asian sea bass, do I have to BLAST the primers to look at similar sequence in Asian seabass or similar sequence in amplifying genes in other sea bass species as well.

The basis of my questions is, since the genome information of non-model organism is limited, is it possible to screen primers for other genes it might amplify in closely related species. This genes might or might not be present in the organism of interest.

2. The PhD student that I working along is using alpha-actin as her RT-control. Is there any review on choosing the correct RT-control? I am currently searching for it but would welcome any reviewed articles by anyone.

Much Obliged,
Jevandrix

#6 Trof

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Posted 05 June 2011 - 10:44 PM

1. It's hard to say when you work with a non model species, but even if the protein sequence is conserved, similarity od cDNA may be even lower. So in my opinion it's not very helpfull to tell you if the primers will be really specific. But if you only have few sequenced genes from your species, that may be the only way how to assess potential nonspecifity.

2. Since RT control only checks if the cDNA is OK, I don't think it matters that much what gene is used, as long as it's constitutively expressed in every sample.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#7 Jevandrix

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Posted 08 June 2011 - 07:51 PM

Dear Ms/Mrs. Trof,

I am having a hard time in understanding this - "On the other hand it's not possible to mark taget and excluded regions, as does primer3 which is sometimes important if you want to design primers in a specific exon or something like that"

Does it mean Primer 3 better than Primer BLAST in marking exon and intron?

I am having trouble in whether to go ahead and use Primer BLAST or other softwares?
I would certainly welcome any software recommendation.

Much Obliged,
Jevandrix

#8 Trof

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Posted 15 June 2011 - 03:05 AM

In primer3, when you paste sequence you can mark target region (region that must be included in the amplicon) with [] brackets and excluded region (where the primers must not be) with <> brackets.
This doesn't work in PrimerBLAST in spite of using the same algorithm to find primers.

Primer3 only accepts pasted sequence, if you want to design intron spanning primers you have to mark the exon-exon junction with [] brackets (you can only mark one particular with this at a time, not all of them). Also if you don't want the primer to be on the polymorphic SNP you put it inside <> mark and primers wouldn't be designed there.

PrimerBLAST can design primers from GeneBank mRNA accession number of the sequence, GeneBank file usually contains information about exons so PrimerBLAST can design intron spanning assay (on any exon-exon junction of the gene), because it knows where all the introns are. Primer3 can't do this.

Sou in you want primers for realtime, intron spanning and you don't care where i the gene they are, you do better with PrimerBLAST. But whenever you need primers avoidnig a particular site or in specified region you do better with primer3.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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