Why I lose my lettuce hairy roots RNA about 5 or 6 hours after extraction with licl method and on electrophoresis only it's DNA band will remain with no RNA?
I do the extraction and I get the RNA bands with a tiny small DNA with that; but after I used DNase and for one of my samples and I run it on gel with my RNA extraction sample wihout DNase;I saw no bands in the sample with Dnase and no RNA bands in the sample without DNase that I had the bands just a few hours ago when I run it on the gwl after extraction for the first time. I don't know why I lost my RNA? I keep all the things on ice too,and keep the sample in -20 at the first hours of extraction,but then keeping it on -40.
and I also want to know what will happen if I use DNase more? Does it effect my RNA Bands?!
and another more question... I did gene tranformation; and I get transgenic hairy roots of lettuce. Can I just do the RTpcr and cDNA synthesis without using DNase?!
Zahra Amiri
My advise, first of all, is try trizol method. Personally I used LiCl method to isolate plant RNA. I faced many problems. I used to get good concentration of RNA in that method though failed to get good results on down stream processing.
1 Why?
DNA is more stable than RNA.
2. DNAse problem
usage of PEG in the isolation process need more care I think. Better use alternative methods.
- Yes you can omit the DNAse step:
- It is not uncommon to lose RNA when you reat with a lot of DNAses as they contain trace amounts of RNAse if purified (as opposed to recombinant DNAse)
- Try and carry out your RNA extraction at room temp and because of your issues buy in a DNAse that is 'certified' as RNAse free: Qiagen for example do one
- You can omit the DNAse step but if for example you then want to use that RNA to measure gene expression by qPCR you need to make sure your primers cross exon exon boundaries:
- That way they will only target mRNA and not trace amounts of genomic DNA not removed by DNAse digestion
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