RT-PCR primer design guide - How to check gene structure and design the primer? (Nov/03/2003 )
Hello everybody!
Am a new member of this forum, am a research student.Can any one of you help me regarding this.I have a gene sequence of lacZ from E coli .I had designed primers for its amplification and it worked now I want to design RTPCR primers and will use total RNA from E coli as template and want to maake cDNA first so how do I design RTPCR primers so that from the cDNA I can later amplify the lacZ gene???Please help me out
Probe library from Exiqon or Roche-applied biosciece is good in finding primers which span an intron
Here is the SOP in our lab, I got an almost 100% (ot of 30 genes I tried) accuracy for rt-pcr. However, there is something like 60-70% success in QRT_PCR without trying any optimization in PCR parameters or concentrations...
Designing Primers for Real Time PCR:
PERL PRIMER is the program of choice...
Download it from: (click this to see documentation..)
http://perlprimer.sourceforge.net/
In realtime PCR primer design, following points should be considered...
1. Primers should encompass an intron to see any genomic contamination.
2. One of the primers should reside on the intron exon boundry, (most of the primer length should reside on the 5' exon to prevent mispriming...)
3. A GC clamp should be present on the 3' most end of the primer to prevent mispriming.
4. The amplicon size is recomended to be between 100-300 bp.
Primer Design Protocole
1. Go to the Entrez Gene database.
2. Find the REFSEQ of your gene.
3. Get the refseq cDNA of your gene...
* There is a link to the REFSEQ db from the ENTREZ GENE entry of the gene...something like NM_XXX....
* Click FASTA format sequence of the REFSEQ cDNA,
* Save the line starting with a greater than ( > ) sign as the identifier of your sequence...
4. Go to BLAT ( http://genome.ucsc.edu/cgi-bin/hgBlat )
5. Get the genomic sequence which corresponds to your gene by pasting your cDNA sequence in to BLAT and clicking submit.
6. Then just click find primers on perl primer, after pasting the genomic and cDNA sequences of the gene.
7. Finally, after you select your primers check the products which might be produced by these primers by epcr tool of NCBI (http://www.ncbi.nlm.nih.gov/sutils/e-pcr/reverse.cgi). Read the help of that tool if it is first time...
* Enter your primers in 4 column format in REVERSE STS search tool: Four-column format, exactly one STS per line, columns are: label for sts, left primer, right primer, and product size (can be two dash-separated numbers for range of sizes).
!!Record primer information with at least the following information in an excel file
* Primer name (Gene symbol, primer number(should be unique in the lab), F or R, e.g: ACTB1F, ACTB2R)
* Target gene
* Primer sequence
* TM of primer
* Unique accession number of the sequence from which primer is designed. (gi number of NCBI is a good option, do not use gene accession numbers or gene symbols, they do not identify a sequence, sequence information for genes are always changing!!!)
* Length of the primer
I got so much from this discussion. thank you all here. But I have a question, has anyone investigated how seriously (to what extent) the NDA contamination affects the RT-PCR results?[/i][/b]
let me add my own opinion about the DNA contamination in RT-PCR.
i am doing Ratio-RT-PCR to investigate the expression of N-cadherin in cardiomyocytes.
this method is to express the mRNA level by presenting its ratio to some reference. here for example, if i have 100 cells, then the gene in DNA of the target should be 200, let 0.1 be the contamination rate (it is not so low). then the DNA contamination in cDNA should be 20. then let estimate the number of cDNA. in a cell, one gene expresses 10 mRNA (is it reasonable?) then we will roughly have 1000 cDNA, so the contamination rate before PCR should be 2%. I do not think this data constitute a big problem for Ratio-RT-PCR. therefore, spanning different exons is not so important for the primer design in Ratio-RT-PCR. is anyone agree with me? or any comments to the above analysis? thank you.
Primer3 plus is one of the most comprehensive primer design software available. It will remove the guessswork involved in primer design. You can also look for rt-pcr primer design tools such as PCR Now -(http://pathogene.swmed.edu/rt_primer/), the pcr suite and qPCR primer design tool -Beacon designer.