1. Select the appropriate amplification fragment for the target gene sequence

Check whether the following three sites have suitable amplified primers , probes and reaction conditions for QRT-PCR .

RTPrimerDB (http://medgen.ugent.be/rtprimerdb), relatively more species

PrimerBank (http://pga.mgh.harvard.edu/primerbank/index.html) person, rat

Real Time PCR Primer Sets (http://) , mouse , rat

If no suitable or has been proven to provide a reference, the following design for reference only:

A. The most widely used commercial software Beacon Designer (http://)

B.DIY Software Prime Express

C. If Beacon Designer can't get the results you need , or if you don't get enough choices for the design , you can choose Sigma-Genosys http://) service plan , detailed and thoughtful

D. A free web-based primer and probe design program : http://

You can select 4-6 pairs of primers to test , and select primers for amplification efficiency and sensitivity . This software can be directly compared with NCBI website , and virtual electronic PCR is performed by NCBI ePCR .

Introduction to Primer Design

DNA primer length : 15-25 bases

GC content : about 50%

If primers are bound to the AT region , consider replacing several bases with LNA , reducing the length of the primers and avoiding primer secondary structures and 3' The effect of the terminal dimer . Due to the competition between the primer and the template and the difference between the probe and the target , intra-hybridization , reverse-repetition, etc., cause the primer-initiated probe to reduce the binding efficiency , thus we chose primer dimer â–³ G is negative, namely: <10 kcal / mol without continuous G / C..

The preservation of primer probes generally follows the following principles :

The forward and reverse primers are stored at -20 degrees and the concentration is 10mM Or 10 × working concentration . The probe should be stored in the dark, stored at -70 degrees , preferably in the lyophilized powder state, the working concentration of the liquid is generally stored for about two weeks.

2. Enter the target sequence and compare it with BLASTn at http://

3. Check the polymorphism of the aligned sequences and possible errors to avoid these regions for primer and probe design.

4. Avoid direct reciprocal regions in the target sequence, and hybridization in the repeat region tends to make the primers non-productively bound, reducing the efficiency of DNA amplification and reducing the sensitivity of the assay.

5. By considering potential splice variants and appropriate required acquisition targets, the boundaries of introns and exons are analyzed by analysis, primarily by cDNA and genomic sequence alignment. It is generally designed to span the longest intron region, which reduces the effects of amplicon contamination by genomic DNA . This is necessary, especially when normalizing with DNA and targeting a specific splice variant. The most economical approach is to have downstream primers span the splice junction, which allows the use of one probe to detect possible splice variants . Then, if the validity and sensitivity are not guaranteed, a design that spans a single exon can be used. We also recommend that the tester treat the sample with DnaseI to remove gDNA contamination.

6. At the RT step, use the (http://) tool to detect the folding of the target sequence at a specific temperature, avoiding some highly secondary structures. Regions, those regions where probes and primers are less efficient to bind

7. Use 60-150 bp amplification products as much as possible, and the GC content is 60 % or slightly smaller to determine efficient denaturation and higher reaction efficiency. High GC sequences are prone to non-specific reactions, and short sequence amplification is a reduction in the likelihood of amplification of gDNA contamination. Short sequences are easily synthesized and used to amplify multiple standard curves. It is best to use reverse transcription of oligodT to design the amplicon located close to the template. 3' area.

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