1) Poor yield of total RNA is mostly due to incomplete sample Lysis, thus leading to incomplete release of RNA. Since good yield and good quality of total RNA are only assured when the sample is properly handled and lysed completely, DO NOT use more than the amount of sample suggested in the protocol.
2) Thorough cellular disruption is critical for high RNA quality and yield. RNA that is trapped in intact cells is often removed with cellular debris and is unavailable for subsequent isolation. Therefore, it is crucial to choose the disruption method best suited to a specific tissue or organism to maximize yield. Mechanical cell disruption techniques include grinding, homogenization, vortexing, sonication etc. Complete disruption of some tissues may require using a combination of these techniques.
3) Another, more common cause of low RNA yield is overloading the column, which can cause the column to clog or can prevent the RNA from binding to the membrane efficiently. Methods that reduce viscosity, such as reducing sample amount, disrupting the sample more extensively, and centrifuging to remove insoluble remains, will increase RNA yield. If yields are still lower than expected, consider diluting the clarified lysate and splitting loading into two columns, which will further reduce the concentration of contaminants and improve RNA binding and recovery.
4) When RNA is to be eluted, make sure that RNase-free ddH2O is added onto the membrane and absorbed completely. If ddH2O still remains on the membrane, pulse centrifuge the column for a few seconds to drag it into the membrane.
Three critical steps, if not performed well can cause RNA degradation. Handling and storing of samples, disruption of samples and storage of eluted RNA. (1) Most animal tissues can be processed fresh (unfrozen). It is important to keep fresh tissue cold and to process it quickly (within 30 minutes) after dissecting. If samples cannot be processed immediately, it should be flash frozen in liquid nitrogen and stored at -80°C. Samples should be handled with RNase-free tools. (2) When the sample is disrupted, disruption needs to be fast and thorough. Slow disruption (e.g. placing cells or tissue in RB Buffer without any additional physical shearing) may result in RNA degradation by endogenous RNase released internally, yet still inaccessible to the protein denaturant in the buffer. (3) After elution of RNA with RNase-free ddH2O, store RNA at -80°C. (4) Degradation of RNA may also occur during loading into a gel. Use gel and fresh running buffer prepared using DEPC-treated ddH2O, as well as a properly cleaned geltray and tank for electrophoresis. Adding EtBr directly into the gel can also avoid possible degradation of RNA that may occur during gel staining.
No, some genomic DNA (and plasmid DNA, if present) can be co-purified with RNA. DNA can be removed by adding RNase-free DNase I to the RNA sample. DNase I can then be removed by phenol/chloroform extraction.
The extra filter columns are effective in clearing cell debris and ensuring full cell Lysis.
For isolation of Cytoplasmic RNA from animal cells or eukaryotic cells, the Total RNA Mini Kit (Blood/Cultured Cell) will satisfy this requirement. Extra buffer (not included in the kit) will have to be prepared to lyse the plasma membrane before proceeding with the regular protocol. Plasma Lysis Buffer Components: (Pre-cool to 4°C) 50 mM Tris-Cl, pH 8.0 140 mM NaCl 1.5 mM MgCl2 0.5% (v/v) Nonidet P-40‡ (1.06 g/ml). Just before use, add: 1,000 U/ml RNase inhibitor 1 mM DTT. Add Buffer to lyse plasma membrane: For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Carefully resuspend cells in 175 µl cold (4°C) Plasma Lysis Buffer, and incubate on ice for 5 minutes.
The Presto™ Mini RNA Yeast Kit.