I am trying to amplify target sequence using FFPE DNA. Initially, I managed to get the target band I want although it is weak. However, when I try to repeat the test using same formula, no band was observed. Has anyone encountered the same problem?
Try using efficient method for extraction for better DNA
QIAamp DNA FFPE Tissue Kit
Rapid purification of high-quality, ready-to-use DNA
Consistent, high yields
Complete removal of contaminants and inhibitors
Source: http://www.qiagen.com/jo/products/catalog/sample-technologies/dna-sample-technologies/genomic-dna/qiaamp-dna-ffpe-tissue-kit
First before extraction
Sure the DNA degradation from tissue sample is one the main cause of PCR failure and gel ectrophoresis can solve this before starting extraction protocol...
-Try best method for efficient fixation with less damaged DNA
The Isolation of Nucleic Acids from Fixed, Paraffin-Embedded Tissues–Which Methods Are Useful When?http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0000537
Fundamental study on the mechanism of DNA degradation in tissues fixed in formaldehyde
http://www.ncbi.nlm.nih.gov/pubmed/2120290
Comparison of Methods for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded Archival Tissues
http://www.medsci.org/v11p0494.htm
Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0077771
2. Extraction procedure efficient kit or methodology for high quality DNA (Spectrophotometer)
3. Post-extraction manipulation of DNA
4. General troubleshooting in PCR reaction
https://www.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide
No band
http://www.fws.gov/aah/PDF/PCRTS1_NoBand.pdf
5. Using positive control for your PCR reaction
Thank you for the all the answers. Salwa, I am using
QIAamp DNA Mini Kit. is it ok? My samples are FFPE from hospital archive, the tissue fixation is out of my control. I am trying the positive control you suggested.
Harald, My target band size is 160 bp, so I guess I should be able to amplify it as it is
Formalin-Fixed tissues are notorious for their degraded DNA. How degraded depends on the details of the fixation procedure. Treating your DNA with ligase to repair single-strand nicks might help, though I don't know if it has ever been tried. Including an internal control such as beta-globin or actin is essential in order to assess the amount of amplifiable DNA in each sample, If you have access to real time PCR, it would probably pay to redesign your PCR. Using SYBR green and a Tm analysis you can get down to about 50 bp. Remember that your internal control should be close in size to, but not smaller than, your target amplicon.
Thank you, Andrew. I just used a positive and negative control alongside with a sample. My positive and negative control work fine but no result was observed for the sample. This means the working solution is ok, so the problem should be the FFPE?
Hi there, I just ran an internal control like Andrew suggested and result shows that there was no target band for samples but has target band for my positive control. It appears that my FFPE samples were degraded through repeating freezing and thawing while optimizing. That was the culprit.
Thank you for all the answers. I have tried the suggestions by all of you. Thank you again for taking time to answer my question.
Good to hear you got to the bottom of the problem. Pity about the samples. Good luck with the rest of your work. Andrew
I am also working on FFPE samples, i had the same problem initially and after that i optimized the PCR conditions and it worked well.Try increasing dNTP and Taq polymerase concentration upto 4 fold or use Ampliqon Red Dye Master Mix.
It is to be noted that, normally the FFPE DNA samples contains PCR inhibitors and to get rid of it try using good extraction kit. I am using Promega's FFPE gDNA miniprep and it is giving good results for me.
Hi, I am finally getting the result I want.
These is what I did:
1. extracting DNA using Qiagen kit (I did not use the one specific for FFPE)
2. dilute my samples before PCR
3. I used hotstart master mix.
Thank you everyone for giving the comments and suggestions.
hii Boon Lee Chai
can you please tell me what is the difference between hotstar master mix and normal master mix?
even i am dealing woth FFPE DNA and facing same problem bands are not coming consistent
should i design my primers less than 250bp?
what protocol should i follow for doing pcr as my downstream application is sanger sequencing...
thank you
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