1. The next gel electrophoresis the weather is suggested:
- use 1X TAE buffer rather of 1X TBE - use agarose gel within the power of 1.1%-1.2% - add ethidium bromide (EtBr) towards the gel and electrophoresis buffer to prevent the extra (potentially RNAse-prone) step of gel staining - always employ fresh gel and buffer in addition to clean electrophoresis equipment for RNA analysis. Put on mitts to safeguard RNA samples from degradation by nucleases and steer clear of a hands connection with EtBr. - use running current as much as 10 V/cm (10V for every cm of space between your electrodes in electrophoretic chamber). Don't use high current to prevent RNA degradation during electrophoresis.
2. Heat an aliquot from the RNA solution at 70C for 1 min and put it on ice before loading on the gel.
3. Load a known quantity of DNA or RNA ladder alongside your RNA sample like a standard for figuring out the RNA concentration. RNA concentration could be roughly believed presuming the efficiency of EtBr incorporation in rRNA is equivalent to for DNA (the ribosomal RNA may be described as a double-stranded molecule because of its extensive secondary structure).
4. The very first manifestation of RNA degradation around the non-denaturing gel is really a slight smear beginning in the rRNA bands and increasing towards the section of shorter fragments. RNA showing this extent of degradation continues to be great for further procedures. However, when the downward smearing is really pronounced the rRNA bands don't have a discernible lower edge, this RNA ought to be discarded.
The next characteristics indicate effective RNA preparation:
- For mammalian total RNA, two intensive bands ought to be observed against an easy smear. These bands represent 28S and 18S rRNA. The number of intensities of those bands ought to be about 1.5-2.5:1. Intact mammalian poly (A)+ RNA seems like a smear sized from .1 to 4-7 (or even more) kb with faint 28S and 18S rRNA bands.
- Within the situation of RNA from non-mammalian sources (plants, insects, yeast, amphibians), the standard mRNA smear around the non-denaturing agarose gel might not exceed 2-3 kb. Furthermore, the overwhelming most of invertebrates have 28s rRNA having a so-known as "hidden break" (Ishikawa, 1977). In certain microorganisms the interaction between your areas of 28s rRNA is quite weak, therefore the total RNA preparation exhibits just one 18s-like rRNA band even on the non-denaturing gel. In other species the 28s rRNA is much more robust, so it's still visible like a second band.
Note: In case your experimental RNA is shorter than expected and/or degraded based on electrophoresis data, prepare fresh RNA after checking the caliber of RNA purification reagents. If problems persist, you may want to find another supply of tissue/cells. In some instances, partly degraded RNA is just available (e.g. tumor samples or hard treated tissues). This RNA can be used as cDNA preparation, nevertheless the cDNA sample contains reduced quantity of full-length molecules.
Total RNA from endothelial human cells.
- Generally, genomic DNA contamination doesn't exceed the quantity located on the agarose/EtBr gel like a weak gang of high molecular weight. Such contamination has no effect on cDNA synthesis. DNase treatment to degrade genomic DNA isn't suggested. In some instances, more than genomic DNA can be taken off by LiCl precipitation or by phenol:chloroform extraction.