why mRNA difficult to isolate - (Jul/01/2005 )
I would like to made cDNA from mRNA. I have some problem with RNA extraction. I extracted RNA from fungi by using QuickPrep micro mRNA purification kit (Amersham Biosciences). I got only genomic DNA from this kit and mRNA can not. I asked to Amersham Biosciences with that problem but no answer. I am sure that i always do follow the manual of kit. Can every one help me?
I try to used mycelium of fungi after 2-8 days cultivation but I can't get mRNA
Why not skip the mRNA step and make cDNA directly from total RNA?
pBluescript is right.
However, when your mRNA amount is too little, you can try to extract total genomic nucleic acid, treated with DNase, purify and proceed to cDNA synthesis.
I already did extract total genomic nucleic acid, treated with RNase free- DNase I.
DNase I in the samples were inactivated by heat at 70 C for 5min. After RT-PCR and electrophoresis, I could not see any band from RT-PCR samples. This mean that did my samples have not mRNA ? RNeasy Plant mini kit from Qiagen was used in extraction.
Maybe you have RNase contamination somewhere. I had RNase contamination once in my cDNA synthesis reagents.
Use a lot of cells for your RNA extraction i have found that with bacteria, few cells gives poor/no result, but making a 50ml culture i now get excellent RNA. mRNA only represents about 5% of total RNA so trying to get mRNA directly could be hard.
Also depending on what you are looking for if you are using a lysis solution bear in mind your cells may be stressed and therefore stress response may be activated 'polluting' your mRNA population. I bead beat my cells in trizol, an RNA isolation solution (invitrogen, available for eukaryote) this way the cells are instantly lysed without a iope stress response and therefore the mRNA population is more representative, a 'snap-shot' if you like.
Hope this helps, but i would definitely encourage a large mass for extraction.