Designing QPCR primers

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderators: honeev, Leonid, amiradm, BioTeam

Post Reply
Newbie
Garter
Garter
Posts: 22
Joined: Wed May 03, 2006 3:21 am

Designing QPCR primers

Post by Newbie » Wed May 03, 2006 3:52 am

Hi everybody,

I am as my name suggests entirely new to molecular biology techniques and designing PCR primers. I was asked to design primers for QPCR using SYBR and I was told to just design it like normal primer which I never had experience doing either. All I know are some basic principles of how primers and PCR work but the actually nitty gritty of things are an entirely different story. What kind of information do I need? I've read about some programs that can design primers for you but what do I do first? They say I need to know the genomic sequence and cDNA, why is that and where do I find it? I tried looking for the sequence and there are so many isoforms, which do I choose? Do i need to look for the open reading frame? I have no idea how to fiddle around with the parameters, do i display the exon and introns or just the exons? Do I need to exclude the stop codon for the reverse primer? Please someone give me some direction!
Last edited by Newbie on Fri May 19, 2006 4:24 pm, edited 1 time in total.

User avatar
LilKim
Coral
Coral
Posts: 436
Joined: Mon Mar 20, 2006 8:36 pm
Contact:

Post by LilKim » Wed May 03, 2006 4:42 am

Hey newbie,

it's would be really difficult for me to help you (or check your primers). Because, you're not giving me enough information to help you design your specific primers here (in this forum).

Are you in a lab?

I STRONGLY suggest that you go back to the person who told you to design primers and ask for help.... or ... ask someone else in the lab for help.

Before you even start ...you're going to have to know ...

1. which isoform...
2. the cDNA sequence for that isoform

It's highly likely that the person who assigned the problem can e-mail you the correct sequence.

As far as primer design programs? i've never used one, i've always used designed the primers by looking at the sequences.

good luck!
- KIM

p.s. go to http://www.wikipedia.com and type in Q-PCR (or QRT-PCR) for some explanations of the proceedure.

User avatar
canalon
Inland Taipan
Inland Taipan
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada

Post by canalon » Wed May 03, 2006 11:47 am

But you will find on internet the free Primer3 that designs primers automatically, and for having used it, they work as predicted.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

Newbie
Garter
Garter
Posts: 22
Joined: Wed May 03, 2006 3:21 am

Post by Newbie » Wed May 03, 2006 3:08 pm

Hi,

Yes, I just joined a lab recently and given this new project to look at the gene expression level of these 3 interacting genes. I have no other information other than the names of the 3 genes he wants to look at and some papers describing what they do. The don't think the person that assigned this to me knows the cDNA sequence. Which is why I'm having trouble figuring out where to begin and where to find these sequences. I think he basically I'm on my own and he wants me to start from scratch. I know about Primer3 but like I said I don't know enough about how to use the program to be able to use it. Do I just find the sequence online and copy and paste it there? Aren't there things I need to consider such as opening reading frames or exons and introns. I have no idea, I'm totally lost. I wish there was some detailed guide to show me step by step how to do this...

User avatar
LilKim
Coral
Coral
Posts: 436
Joined: Mon Mar 20, 2006 8:36 pm
Contact:

Post by LilKim » Wed May 03, 2006 7:21 pm

Have you asked the guy in your lab for help? Is there anyone elsein the lab to help you?

Don't be affraid to ask for help, they won't think that you're stupid for needing help (especially if you've never done this before) Besides, asking for help and talking with your fellow lab-people isthe best way to build good, strong relationships. (And i'm sure that someone would be more than happy to help you). Furthermore, if you try to do this on your own... not knowing what you're doing and totally screw-it up... you'll probably look more-foolish that if you just asked for help in the first place.

I'm totally not trying to lecture you or anythin like that... just trying to give you some GOOD advice.

As far as looking up the cDNA sequence.. you can find it on pub med.

go to http://www.pubmed.com and click on "nucleotide" (in the black bar at the top of the page). Then type in the name of your gene (for example: Fmr1).. then check to make sure that it's the cDNA sequence of the correct organism (Fmr1 in homo sapiens). Once you click on an acession number scroll to the bottom... you'll see a cDNA sequence as well as an amino acid sequence.

REMEMBER!!! these databases are NOT 100% correct and it is not uncommon that primers derived from these sequences won't work .... Thus it is imperative that you double check EVERYTHING with the person that assigned you the project. They'd probably be more annoyed if you designed incorrect primers based on an incorrect sequence... than you just asking them for it in the first place!!!

The lab may even be using a cDNA sequence other that what's actually published too .... (This has happened to me in the last 2 labs I worked in ... I used cDNA sequences from my lab... because they were DIFFERENT from what is 'published')

good luck!
- kim

p.s. if your dealing with cDNA you don't have to worry about exons and introns. However, if you're going to be expressing the cDNA you'll need to worry about amplifying "in-frame" for ligation into a plasmid, shine delgarmo sequence.

Newbie
Garter
Garter
Posts: 22
Joined: Wed May 03, 2006 3:21 am

Post by Newbie » Wed May 03, 2006 10:02 pm

Hi kim,

Thanks for the advice. Everyone in the lab seems to be super busy and expect me to figure things out for myself I guess. They just told me to use this website http://www.genome.ucsc.edu to look up the sequence. The problem is I don't know how to play around with the parameters. It gives us the genomic sequence or mRNA sequence. I clicked on genomic sequence that displays both the exons and introns but there is an option that lets us just display the exons which is why I'm wondering if I would need to just display the exons (since introns are non-coding) and design primers from that. I don't need do cloning with the genes, just checking expression levels with QPCR. I guess I should ask for some advice, it just seems designing primers seem so basic and simple to people in the lab that I feel like maybe it's something I should figure out for myself? I don't want to seem like a bother. I think everyone is just so used to designing primers they don't realize I may not know do it. I'm sure it's simple once I learn how to do it...but figuring it out can be a frustrating process when I have never done it before, there are a lot more things to consider than I thought.

User avatar
LilKim
Coral
Coral
Posts: 436
Joined: Mon Mar 20, 2006 8:36 pm
Contact:

Post by LilKim » Thu May 04, 2006 2:54 pm

So i'm assuming that you're going to make cDNA by using a RT reaction.... (so you can go from RNA to DNA)

So you would prime with a poly DT primer for your RT reaction.

After that reaction you'll have cDNA ... And if I were to guess I would say that you'd design your qPCR primers based on the cDNA sequence. (derived from your mRNA transcript)

Of couse... I could be completely wrong!

What i think yyou should do (and what I've done in the past) is designed the primers and then asked a lab person to check them for you.

Ok ok.. I admit!!! I've never made qPCR primers, so I'd just HAVE to ask someone.

I still think you should kinda swallow your pride and stop someone ...and ask them to help you (that's what i'd do, if I were in your situation ... even if they appeared to be "super-busy")

good luck
- kim

Newbie
Garter
Garter
Posts: 22
Joined: Wed May 03, 2006 3:21 am

Post by Newbie » Tue May 09, 2006 4:12 pm

Thanks for the encouragment kim!

User avatar
LilKim
Coral
Coral
Posts: 436
Joined: Mon Mar 20, 2006 8:36 pm
Contact:

Post by LilKim » Tue May 09, 2006 6:33 pm

No problemmo!!! I'm guessing that things worked out! Glad to hear... and good luck with your experiments!
- KIM

Newbie
Garter
Garter
Posts: 22
Joined: Wed May 03, 2006 3:21 am

Post by Newbie » Thu May 11, 2006 6:40 pm

Hi kim,

Well I designed my primers and ordered them...I'll just have to cross my fingers to see if they work...I'm not quite sure what template to use, am I supposed to order them too? well one step at a time I guess...I'm supposed to pop the big question tomorrow...asking the boss to accept me permanently into the lab...I feel like I'm proposing...then I'll be "married" to the lab for the new few years...

User avatar
LilKim
Coral
Coral
Posts: 436
Joined: Mon Mar 20, 2006 8:36 pm
Contact:

Post by LilKim » Thu May 11, 2006 10:02 pm

You template DNA or cDNA is what your qPCR primers anneal to.

So... you'll most likely be extracting DNA (from cells or organs etc. ) or you may be using frozen pre-extracted DNA samples that your lab has somewhere. You'll have to ask the lab people for that stuff.

good luck with "popping the question"

take care!
- KIM

blacklisted
Garter
Garter
Posts: 1
Joined: Tue Jan 06, 2009 10:19 am

Re: Designing QPCR primers

Post by blacklisted » Tue Jan 06, 2009 10:39 am

Hi,
I'm a masters student who has just entered the field of mol bio after doing my under grad in chemistry. I'm kinda clueless about most things around me, but there's one particular question thats bothering me.

I'm going to do qPCRs, and i've realised that the amplicon length should not exceed 250 bp. But i dont see why that should be a problem since a normal PCR can have much larger amplicons. Has it anything to do with the fluorescent dye?

Post Reply

Who is online

Users browsing this forum: Bing [Bot] and 7 guests