Hi,
So I am interested in a way for generating DNA, smallRNA and mRNA from the same biological sample for NGS (ExomeSeq, smallRNAseq and RNAseq). Essentially I am looking for a protocol/kit that gives me 3 extracts for the desired nucleotide fractions.
I read about http://www.qiagen.com/us/products/catalog/sample-technologies/rna-sample-technologies/dna-rna-protein/allprep-dnarnamirna-universal-kit/#productdetails and it sounds interesting, but it gives only one RNA fraction.
Thanks,
I used Macherey-Nagel kit for plant tissues! It is more or less the same as Qiagen. But it also gives you only one fraction. You can check their protocol also online!
Hi
This may be too elementary, but we use Trizol and Qiagen RNeasy kits. QIagen makes columns that are specific for micro RNA. You would take the 'aqueous fraction' above the phenol-chloroform and then add isopropanol and transfer into the column. We use RNeasy micro columns for total RNA (probably loses some miRNA), but they work pretty well RIN usually above 9.5. I had planned on precipitation if we needed the miRNA, we have a speedvac and it is kind of hard to mess up. Good luck!!
There may be several alternative possibilities. For example, when using mirVana miRNA Isolation Kit you can obtain fractions of DNA, RNA and protein. As regards RNA, you may follow the protocol for obtaining total RNA (incl. miRNAs), or small RNAs enriched fraction. Or, you can use total RNA and further separate small RNA fraction by flashPAGE Fractionator System. Or, you can use All-in-One Purification Kit by Norgen Biotek, giving Total RNA, microRNA, total proteins and genomic DNA.
I hope this helps.
Ludek
Lexogen offers the SPLIT RNA Extraction Kit
http://www.lexogen.com/products/split-rna-extraction.html
which does exactly what you want: it splits the extracted RNA at a threshold of ~ 150 nt into a large RNA fraction and a small RNA fraction (including miRNA). Both fractions are purified on silica columns and of highest quality (RIN of 10 possible, depending on input), suitable for RT-PCR, RNA-Seq etc.
The SPLIT Kit includes an acidic phenol extraction step to exclude gDNA from the RNA sample. Recovery of this gDNA from the organic phase is described in
https://www.researchgate.net/post/DNA_extraction_from_TRIZOL_sample
Best,
Lukas
http://www.lexogen.com/products/split-rna-extraction.html
https://www.researchgate.net/post/DNA_extraction_from_TRIZOL_sample
Looks like you have plenty of suggestions to choose from but I'll throw in 2 others. Qiagen's AllPrep and LifeTech's RecoverAll. I've adapted both with good success but what I found was the AllPrep kit yielded more and higher quality DNA and the RecoverAll was better for RNA.
RecoverAll Multisample DNA/RNA is an optimized protocol to isolate gDNA and RNA (including small RNAs) from the same sample in separate eluates. Isolated samples have been extensively tested with qPCR and NGS. Follow the link here: http://www.lifetechnologies.com/order/catalog/product/AM1975?ICID=search-product and click on the user bulletin for the protocol.
What type of samples are you wanting to isolate from?
I would employ Trizol for the extraction of nucleic acids and for microRNAs would have two alternatives mirVana or miRNeasy
http://www.mn-net.com/Products/DNAandRNApurification/RNA/NucleoBondRNADNA/tabid/1325/language/en-US/Default.aspx
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