Normally I use QIAGEN kit for extractions from tumor tissues, but now I am planning to do it from premalignant lesions where I need only epithelial cells.
I tried taking serial cryo sections and processed them with same Kit, but it did not work, maybe because of quantity of tissue. Can anyone suggest a method to extract DNA and RNA simultaneously and in enough quantity so that I can use it for microarray and further experiments?
We have used Trizol followed by RNAeasy kit for RNA purification for microarray analysis. You can also extract DNA from the Trizol fraction.
We've used Epicentre kits for Buccal epithelial swabs and they work well. Machery-Nagel do a range of kits which are cost effective and these can be bought direct or from Fisher Scientific.
if your fragments are enough big, you can use the extraction with Phenol-Guanidine.Otherwisw you can try with trizol method! Good luck.
I can recommend you to try kits from Epicentre . I use kits from Epicentre to isolate DNA and RNA (MasterPure Complete DNA und RNA Purification Kit) from aphids and leafhoppers and the downstream application works well!
We have used Trizol followed by RNAeasy kit for RNA purification for microarray analysis. You can also extract DNA from the Trizol fraction.
We've used Epicentre kits for Buccal epithelial swabs and they work well. Machery-Nagel do a range of kits which are cost effective and these can be bought direct or from Fisher Scientific.
For RNA
Pick up your samples and place in Qiazol (Qiagen). Vortex the Qiazol mix at room temperature for 5 min and then pipette up and down to facilitate disruption. Subsequent purification step: Add 150 μl of chloroform (sigma) to the Lysates, previously thaw at room temperature, and vigorously shake during 15 seconds. After 3 minutes of incubation at room temperature centrifuge the homogenates at 6000 rpm during 15 min. Recover the aqueous phase and purify the total RNA using RNeasy (Qiagen) following the manufacturers recommendations.
You could try to first prepare suspension of mononuclear cells by for example collagenase digestion method, then sort out the cells of interest by FACS (cytokeratin-specific antibody might be suitable for epithelial cells) and then extract RNA/DNA by method of your preference. Good luck, KS.
You can use this machine called Maxwell® 16 MDx Research System for your extraction. check this link for details http://www.promega.com/products/instruments/maxwell-16-instruments?gclid=CNzMudiAibMCFYXLtAodSWUAIw
Thanks all for ur suggessions, I am working with human buccal epithelium, and planing to do simultaneous DNA -RNA extraction, I think QIAGEN micro kit gives very low yield and as i am working with patient samples so i have to optimally use it .
Why don't you try DNA extraction kit for Tissue from Qiagen. The are good enough and save lot of time. I use them for my samples in my lab.
Priyanka you must be using DNA extraction kit, try the same but the different one which comes specially for tissue from Qiagen.
Or else try kit from Macherey Nagel. This was the company who designed DNA extraction for Qiagen, now the provide their own kit.
all the best
If you are working with Buccal swabs there is an Epicentre kit designed to allow RNA and DNA purification. http://www.epibio.com/item.asp?ID=313
I'd say Trizol reagent and then RNAeasy. You might need to pre-digest epithelium if a lot of ECM, though.
for low DNA /RNA samples I use Allprep DNA/RNA extraction micro from Qiagen. I tried a lot of kits but this one is really good. DNA is good quality for PCR with as few as 100 cells and 1 copy gene. RNA is amplified using life tech HS RNA amplification kit for hybridization.
I extract RNA by Qiagen's RNeasy plus mini kit. For DNA extraction from same sample, I add 100ul more lysis buffer (There is extra in the kit). After first spin, I save the bottom sticky 100ul for DNA, then extract DNA by Promega's Wizard SV gDNA column kit by adding 500ul Promega's lysis buffer in their kit (There is extra in the kit also).
For RNA from human cells, I've had great success with the Nucleospin RNA kit from Macherey Nagel.
I used "Simultaneous extraction of DNA and RNA using Qiagen ALLPrep DNA/RNA minikit # 80204"
Cheers
Lara
I'm extracting RNA using TRI reagent (it smells awful, so work in aerated laminar box !!!) Result, yelds are very good!
I should clarify that I work with cell cultures (preadipocytes), so may be it depends on the cell type!
I agree with collegs about TRIzol (in fact, it is almost the same)!
I guess the problem is more on how you collect and stabilize your sample at the point of collection until you process it, especially for RNA samples. If your swabs (post collection) sets in room temperature over 2 hrs, you RNA will be hard to get with any kit or extraction chemistry. We have recently developed a swab-based collection system that can stabilize RNA from buccal cells for extended periods of time, regardless of transport transit time. Our ISWAB-RNA system we developed is a method of releasing the collected buccal cells from multiple swabs (4swabs/iSWAB device) where the swab is left behind. Allowing for concentrating the multiple swabs content of buccal cells from a subject in a single tube.
http://mawidna.com/products/iswab-rna-collection-kit/
https://www.youtube.com/channel/UCzP6-9obn37wQEmXAVt0f8A
You can find compatible extraction chemistries with ISWAB-RNA from the link below:
https://www.dropbox.com/s/vaz9cxm8ukssgyy/Mawi%20iSWAB%20RNA%20%26%20CELLS-%20RNA%20Extraction%20Protocols.pdf?dl=0
Good luck!
For small amount of tissue I successfully used Qiagen RNeasy MICRO kit (although for RNA only). If you need more yield of RNA you can consider using some pre-amplification kit (e.g. from Applied Biosystems).
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